Combination of Anti-CD40 and Anti-CD40L Antibodies as Co-Stimulation Blockade in Preclinical Cardiac Xenotransplantation

Abstract

The blockade of the CD40/CD40L immune checkpoint is considered essential for cardiac xenotransplantation. However, it is still unclear which single antibody directed against CD40 or CD40L (CD154), or which combination of antibodies, is better at preventing organ rejection. For example, the high doses of antibody administered in previous experiments might not be feasible for the treatment of humans, while thrombotic side effects were described for first-generation anti-CD40L antibodies. To address these issues, we conducted six orthotopic pig-to-baboon cardiac xenotransplantation experiments, combining a chimeric anti-CD40 antibody with an investigational long-acting PASylated anti-CD40L Fab fragment. The combination therapy effectively resulted in animal survival with a rate comparable to a previous study that utilized anti-CD40 monotherapy. Importantly, no incidence of thromboembolic events associated with the administration of the anti-CD40L PAS-Fab was observed. Two experiments failed early because of technical reasons, two were terminated deliberately after 90 days with the baboons in excellent condition and two were extended to 120 and 170 days, respectively. Unexpectedly, and despite the absence of any clinical signs, histopathology revealed fungal infections in all four recipients. This study provides, for the first time, insights into a combination therapy with anti-CD40/anti-CD40L antibodies to block this immune checkpoint.

Full text

Citation: Bender, M.; Abicht, J.-M.;
Reichart, B.; Neumann, E.; Radan, J.;
Mokelke, M.; Buttgereit, I.; Leuschen,
M.; Wall, F.; Michel, S.; et al.
Combination of Anti-CD40 and
Anti-CD40L Antibodies as
Co-Stimulation Blockade in Preclinical
Cardiac Xenotransplantation.
Biomedicines 2024,12, 1927. https://
doi.org/10.3390/biomedicines
12081927
Academic Editor: Andrea Székely
Received: 20 June 2024
Revised: 8 August 2024
Accepted: 9 August 2024
Published: 22 August 2024
Copyright: © 2024 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
biomedicines
Article
Combination of Anti-CD40 and Anti-CD40L Antibodies as
Co-Stimulation Blockade in Preclinical Cardiac
Xenotransplantation
Martin Bender
1,
* , Jan-Michael Abicht
1
, Bruno Reichart
2
, Elisabeth Neumann
2
, Julia Radan
2
, Maren Mokelke
2
,
Ines Buttgereit 1, Maria Leuschen 2, Felicia Wall 2, Sebastian Michel 3,4, Reinhard Ellgass 3, Stig Steen 5,
Audrius Paskevicius 5, Andreas Lange 6, Barbara Kessler 6, Elisabeth Kemter 6, Nikolai Klymiuk 6,
Joachim Denner 7, Antonia W. Godehardt 8, Ralf R. Tönjes 8, Jonathan M. Burgmann 9,
Constança Figueiredo
9
, Anastasia Milusev
10, 11
, Valentina Zollet
10, 11
, Neda Salimi-Afjani
10, 11
, Alain Despont
10
,
Robert Rieben 10 , Stephan Ledderose 12, Christoph Walz 12, Christian Hagl 3,4, David Ayares 13,
Eckhard Wolf 6,14,15 , Michael Schmoeckel 3, Paolo Brenner 3, Uli Binder 16 , Michaela Gebauer 16,
Arne Skerra 17 and Matthias Längin 1
1Department of Anaesthesiology, University Hospital, LMU Munich, 81377 Munich, Germany
2Transregional Collaborative Research Center 127, Walter Brendel Centre of Experimental Medicine, LMU
Munich, 81377 Munich, Germany
3Department of Cardiac Surgery, University Hospital, LMU Munich, 81377 Munich, Germany
4Munich Heart Alliance, German Center for Cardiovascular Research (DZHK), 81377 Munich, Germany
5
Department of Cardiothoracic Surgery, Lund University and Skåne University Hospital, 22242 Lund, Sweden
6Institute of Molecular Animal Breeding and Biotechnology, Gene Center, and Department of Veterinary
Sciences, LMU Munich, 81377 Munich, Germany
7Institute of Virology, Free University Berlin, 14163 Berlin, Germany
8Division of Haematology, Cell and Gene Therapy, Paul-Ehrlich-Institut, 63225 Langen, Germany
9Institute of Transfusion Medicine and Transplant Engineering, Hannover Medical School,
30625 Hannover, Germany
10 Department for BioMedical Research (DBMR), University of Bern, 3008 Bern, Switzerland
11 Graduate School for Cellular and Biomedical Sciences (GCB), University of Bern, 3008 Bern, Switzerland
12 Institute of Pathology, Faculty of Medicine, LMU Munich, 81377 Munich, Germany
13 Revivicor, Blacksburg, VA 24060, USA
14 Center for Innovative Medical Models (CiMM), LMU Munich, 81377 Munich, Germany
15 Interfaculty Center for Endocrine and Cardiovascular Disease Network Modelling and Clinical
Transfer (ICONLMU), LMU Munich, 81377 Munich, Germany
16 XL-protein GmbH, 85354 Freising, Germany
17 Chair of Biological Chemistry, School of Life Sciences, Technical University of Munich,
85354 Freising, Germany
*Correspondence: Martin.Bender@med.uni-muenchen.de; Tel.: +49-89-2180-76507
Abstract: The blockade of the CD40/CD40L immune checkpoint is considered essential for cardiac
xenotransplantation. However, it is still unclear which single antibody directed against CD40 or
CD40L (CD154), or which combination of antibodies, is better at preventing organ rejection. For
example, the high doses of antibody administered in previous experiments might not be feasible
for the treatment of humans, while thrombotic side effects were described for first-generation anti-
CD40L antibodies. To address these issues, we conducted six orthotopic pig-to-baboon cardiac
xenotransplantation experiments, combining a chimeric anti-CD40 antibody with an investigational
long-acting PASylated anti-CD40L Fab fragment. The combination therapy effectively resulted in
animal survival with a rate comparable to a previous study that utilized anti-CD40 monotherapy.
Importantly, no incidence of thromboembolic events associated with the administration of the anti-
CD40L PAS-Fab was observed. Two experiments failed early because of technical reasons, two
were terminated deliberately after 90 days with the baboons in excellent condition and two were
extended to 120 and 170 days, respectively. Unexpectedly, and despite the absence of any clinical
signs, histopathology revealed fungal infections in all four recipients. This study provides, for the
first time, insights into a combination therapy with anti-CD40/anti-CD40L antibodies to block this
immune checkpoint.
Biomedicines 2024,12, 1927. https://doi.org/10.3390/biomedicines12081927 https://www.mdpi.com/journal/biomedicines

Biomedicines 2024,12, 1927 2 of 21
Keywords: CD40/CD40L; co-stimulation blockade; heart; orthotopic heart transplantation;
xenotransplantation
1. Introduction
Cardiac xenotransplantation has seen notable improvement in the last few years and
is becoming the most promising alternative to human heart allotransplantation [
1
,
2
]. In
life-supporting pig-to-baboon experiments, consistent survival for up to nine months was
achieved [
3
–
5
]. A cornerstone for cardiac xenotransplantation is the effective co-stimulation
blockade of the CD40/CD40 ligand (CD40L alias CD154) pathway [
1
,
6
], which plays a piv-
otal role in both T cell-driven inflammation and humoral immune responses [
7
]. In 2022, for
the first time, a 10-fold genetically modified porcine heart was transplanted into a human
as an individual medical treatment after receiving approval by the United States Food and
Drug Administration (FDA) and ethics committee [
8
]. The patient survived for
two months
with an immunosuppressive therapy based on the blockade of the CD40/CD40L
axis [8,9].
As testing of the pig organ donor revealed later, porcine cytomegalovirus/porcine rose-
olovirus (PCMV/PRV) was transmitted to this patient and could have contributed to his
early death [8].
As members of the tumor necrosis factor (TNF) receptor superfamily, CD40L and
its receptor CD40 represent a key “immune checkpoint” [
7
]. However, the expression of
CD40 and its counterpart CD40L differ depending on the type of immune cell [
10
–
13
].
CD40 is constitutively expressed on antigen-presenting cells such as B cells, dendritic
cells, macrophages, monocytes, platelets, fibroblasts and epithelial as well as endothelial
cells [
10
–
12
]. CD40L, on the other hand, is found on activated B and, in particular, T cells
as well as platelets, and its expression can also be induced on monocytic cells, natural killer
cells, mast cells and basophils [10,13].
Since many biological processes like humoral and cellular immunity, tissue inflam-
mation, thrombosis, hematopoiesis and tumor cell fate are regulated by CD40/CD40L
interaction [
7
], both cell surface receptors are under intensive investigation with regard to
therapeutic intervention [
7
,
10
]. Indeed, the blockade of the CD40/CD40L pathway with
either anti-CD40 or anti-CD40L monoclonal antibodies (Mabs) has demonstrated excellent
therapeutic potential in preclinical non-human primate xenotransplantation models up to
now [
3
–
5
,
14
–
17
]. However, despite such successful studies, it is still not known which Mab
type in which dosage will be clinically preferable, and none of these Mabs has received
clinical approval yet [10].
In this context, the initially promising clinical development of an anti-CD40L-specific
therapy [
18
] has been challenged by the finding that the corresponding Mab hu5C8 (ru-
plizumab) was associated with an increased risk of thromboembolic events [
16
]. These
side effects were later shown to be caused by the Fc region of the antibody, which acti-
vates platelets via an Fc receptor-dependent mechanism [
19
,
20
]. Interestingly, in some
experimental studies the blockade of CD40L after both allo- and xenotransplantation was
demonstrated to be more effective in prolonging graft survival compared to CD40 block-
ade [
21
–
23
]. Moreover, mounting evidence over the past years raised the possibility that
the biology behind CD40/CD40L is more complicated than anticipated, such that block-
ade of CD40 versus blockade of CD40L is not mechanistically equivalent [
24
,
25
]. While
CD40L—which
also exists in a soluble form (sCD40L)—was believed for a long time to
interact exclusively with CD40, and all the biological functions were attributed to this inter-
action, newer findings revealed the existence and functional role of alternative receptors,
with some of them also involved in controlling the immune response [22,25–28].
Furthermore, apart from their mutual interaction as part of the co-stimulatory sig-
naling cascade [
29
] both proteins, in particular CD40L, exert distinct autocrine functions
on their respective host cells [
30
]. In fact, the receptor CD40 itself shows homotypic as-
sociation, and cognate Mabs may have agonistic, antagonistic as well as CD40L-blocking

Biomedicines 2024,12, 1927 3 of 21
activity [
31
,
32
], depending on their epitopes recognized. Therefore, there should be a
difference between targeting either CD40 or CD40L with a potent Mab—in addition to the
effects of epitope specificity—in order to achieve an efficient co-stimulation blockade for
xenotransplantation. Beyond that, a combination of both types of antibodies may even
have a synergistic effect.
To elucidate this notion, we used a combination therapy of a well-studied mouse/rhesus
chimeric anti-CD40 IgG4 monoclonal antibody (anti-CD40 Mab) [
11
,
31
] with a humanized
PASylated anti-CD40L antigen-binding fragment (anti-CD40L PAS-Fab) [
3
,
33
] derived
from ruplizumab—which lacks Fc immune effector functions and antigen dimerization
activity but has a strongly extended plasma half-life—as part of the immunosuppressive
regimen for orthotopic pig-to-baboon cardiac xenotransplantation experiments. With this
combination therapy, we aimed at potential synergistic effects of a combined CD40/CD40L
co-stimulation blockade, while at the same time, avoiding the disadvantages of high-dose
anti-CD40 or anti-CD40L monotherapies.
2. Materials and Methods
2.1. Animals
Hearts from six genetically modified piglets were transplanted into male captive-bred
baboons. The piglets (German Landrace/Large White; blood group 0) were homozygous for
α
1,3-galactosyltransferase knockout (GGTA1-KO) and hemizygous transgenic for human
CD46 (hCD46) and human thrombomodulin (hTBM) (Revivicor, Blacksburg, VA, USA and
Institute of Molecular Animal Breeding and Biotechnology, Gene Center, LMU Munich,
Munich, Germany). Six baboons (Papio anubis and Papio hamadryas; blood group A (n= 4)
or AB (n= 2); German Primate Centre DPZ, Göttingen, Germany) served as recipients. The
expression of hCD46 and hTBM was verified post-mortem by immunohistochemistry.
The study was approved by the Government of Upper Bavaria (Regierung von Ober-
bayern (ROB); Munich, Germany). All animals were cared for and treated in accordance
with the Guide for the Care and Use of Laboratory Animals (German Legislation for the
Welfare of Laboratory Animals and US National Institutes of Health).
2.2. Virological Screening
Both the donor pigs and the baboon recipients were screened for PCMV/PRV using
real-time PCR, nested-PCR and immunological methods like Western blot and peptide
ELISA. The procedures were performed as described in parallel at the Free University
Berlin [
34
] and the Paul-Ehrlich-Institute [
35
]. In addition, the donor pigs were tested
for porcine circovirus 3 (PCV3), which was once transmitted to baboon recipients of pig
hearts [
36
], as well as porcine lymphotropic herpesvirus-3 (PLHV-3) using PCR methods as
described elsewhere [34].
2.3. Anesthesia, Surgical Procedures and Heart Preservation
After sedation, induction of anesthesia and endotracheal intubation of the animals [
37
],
surgery was conducted as published in detail elsewhere [3].
In brief, after median sternotomy of the donor animal, the aorta was cross-clamped
and antegrade non-ischemic preservation commenced immediately; continuous perfusion
with 8
◦
C oxygenated, hyperoncotic solution containing albumin, hormones, nutrients
and erythrocytes [
38
,
39
] was provided by an extracorporeal heart preservation system
(University of Lund, Lund, Sweden) consisting of a pressure- and flow-controlled roller
pump, an O
2
/CO
2
exchanger, a leukocyte filter and a cooler/heater unit. During storage,
the heart was preserved the same way and the perfusion pressure kept at 20 mmHg.
After median sternotomy in the baboon recipient, extracorporeal circulation was
installed and started. Explantation of the recipient
´
s native heart and xenotransplantation
followed the techniques of Lower and Shumway [
40
]. The donor heart was intermittently
perfused for 2 min every 15 min during implantation.

Biomedicines 2024,12, 1927 4 of 21
2.4. Immunosuppression
For induction therapy, all animals received the B cell-depleting anti-CD20 Mab rit-
uximab (Mabthera; Roche Pharma, Basel, Switzerland), ATG (thymoglobulin; Sanofi,
Paris, France) and a combination of an anti-CD40 Mab (50 mg/kg body weight (bw);
mouse/rhesus chimeric IgG4 clone 2C10R4, NIH Non-human Primate Reagent Resource;
Mass Biologicals, Boston, MA, USA; courtesy of K. Reimann) and the anti-CD40L PAS-Fab
(20 mg/kg bw; XL-protein, Freising, Germany).
Immunosuppression was maintained with mycophenolate mofetil (CellCept; Roche
Pharma), methylprednisolone (urbasone soluble; Sanofi) and the combination therapy from
above comprising anti-CD40 Mab (50 mg/kg bw once weekly, reduced to 30 mg/kg bw
after 1 month) and anti-CD40L PAS-Fab (20 mg/kg bw every 4 days, reduced to 10 mg/kg
bw after 2 months) at successively decreased doses (Figure 1).
In addition, all animals received a therapeutic regimen to slow down xenograft
overgrowth which was described in detail elsewhere [
3
,
4
]. Methylprednisolone was ta-
pered down quickly and additional antihypertensive drugs (enalapril (Enahexal; Hexal,
Holzkirchen, Germany) and metoprolol tartrate (Beloc; AstraZeneca, Cambridge, United
Kingdom)) as well as the mTOR inhibitor temsirolimus (Torisel; Pfizer, New York, NY,
USA) were administered.
2.5. Laboratory Tests
Blood samples were taken from baboon recipients prior to xenotransplantation, regu-
larly during each experiment and before euthanasia. Effusion specimens were collected by
thoracocentesis. Measurements were performed by the Institute of Laboratory Medicine
(University Hospital, LMU Munich, Munich, Germany).
2.6. Assessment of Non-Gal-α(1,3)Gal Xenoreactive Antibody Levels
Plasma levels of baboon IgM and IgG directed against cellular antigens of Gal-
α
(1,3)-
Gal-deficient pigs were measured by flow cytometry following the consensus protocol
published previously [
41
]. In brief, GGTA1-KO,hCD46/hTBM transgenic porcine aortic
endothelial cells (PAEC) were collected and suspended at 2
×
10
6
cells per ml in staining
buffer (PBS-1% BSA). Plasma samples were heat-inactivated at 56
◦
C for 30 min and diluted
1:20 in staining buffer. PAEC were incubated with diluted baboon plasma for 15 min at
37 ◦C.
Cells were then washed with cold staining buffer and incubated with goat anti-
human IgM-PE (SouthernBio, CAT: 2020-09; SouthernBiotech, Birmingham, AL, USA) or
goat anti-human IgG-FITC (Invitrogen, Ref: 62-8411; Thermo Fisher Scientific, Waltham,
MA, USA) for 1 h at 4
◦
C. After rewashing with cold staining buffer, cells were resuspended
in PBS, fluorescence was acquired on FACS LSRII (BD Biosciences, Franklin Lakes, NJ, USA)
and data were analyzed using FlowJo analysis software for detection of mean fluorescence
intensity (MFI) in the FITC channel or in the PE channel. Data were then plotted using
Prism 9 (GraphPad, San Diego, CA, USA).
2.7. Cytokine Secretion Profile
Systemic cytokine and chemokine levels (Eotaxin, IL-6, IL-12p70, I-TAC, MCP-1,
MIG and TNF-
α
) were measured in sera and pleural effusions of the animals prior to
xenotransplantation or at different timepoints after xenotransplantation. For cytokine
and chemokine detection, an Invitrogen™ ProcartaPlex™ NHP Cytokine & Chemokine
Panel, 29 plex assay (ThermoFisher Scientific) and a Luminex
®
100/200 analyzer (Luminex
Corporation, Austin, TX, USA) were used. Standards and samples were prepared according
to the manufacturer’s instructions and cytokine concentrations were calculated by Xponent
software version 3.1 (Luminex Corporation).
2.8. Necropsy and Histology
Necropsies and histological analyses were performed at the Institute of Pathology
(Faculty of Medicine, LMU Munich, Munich, Germany). Specimens were fixed in formalin,

Biomedicines 2024,12, 1927 5 of 21
embedded in paraffin and plastic, sectioned and stained with hematoxylin and eosin
using standard procedures. Grocott methenamine silver staining was performed for better
visualization of fungal structures.
2.9. Immunohistochemical Staining
Myocardial tissue was fixed with 4% formalin overnight, paraffin-embedded and
3µm
sections were cut and dried. Heat-induced antigen retrieval was performed in Target
Retrieval solution (DAKO S1699, Agilent Technologies, Santa Clara, CA, USA) in a boiling
water bath for 20 min for detection of hTBM and in citrate buffer, pH 6.0, in a steamer
for 45 min for detection of hTBM, respectively. Immunohistochemistry was performed
using the following primary antibodies: mouse anti-human CD46 monoclonal antibody
(HM2103, Hycult Biotech, Uden, The Netherlands) and mouse anti-human thrombomod-
ulin monoclonal antibody (sc-13164, Santa Cruz Biotechnology, Dallas, TX, USA). The
secondary antibody was a biotinylated AffiniPure goat anti-mouse IgG (115-065-146, Jack-
son ImmunoResearch, West Grove, PA, USA). Immunoreactivity was visualized using 3,3-
diaminobenzidine tetrahydrochloride dihydrate (DAB; brown color). Nuclear counterstain-
ing was performed with hemalum (blue color). In addition, routine immunohistochemical
analysis for complement component C4d (C4d Polyclonal Antibody, Invitrogen/Thermo
Fisher Scientific) was performed on all specimens.
2.10. Immunofluorescence Staining
Myocardial tissue samples were embedded in TissueTek O.C.T compound (Sakura 4583,
Sakura Finetek, Torrance, CA, USA), and 10
µ
m thick sections were fixed and permeabi-
lized with ice-cold 1:1 acetone (141007.1211, AppliChem, Darmstadt, Germany)/methanol
(1.06009.2500, Merck Millipore, Darmstadt, Germany) for 10 min at room temperature
and rehydrated in TBS for 5 min. After blocking for one hour at room temperature with
TBS-3%BSA (Merck A7030, Merck Millipore), cryosections were incubated overnight at 4
◦
C
with anti-porcine CD31 antibody (MAB33871, R&D Systems, Minneapolis, MN, USA), mon-
oclonal anti-complement component C5b (InvitrogenDIA 011-01-02, clone aE11, Thermo
Fisher Scientific), anti-human C3b/c (DAKO A0062, Agilent Technologies), anti-human
C4b/c (DAKO F0169, Agilent Technologies), anti-human IgM (Sigma F 5384, Merck Mil-
lipore), polyclonal anti-human fibrinogen (DAKO F0111, Agilent Technologies), wheat
germ agglutinin (WGA from triticum vulgaris L4895 Sigma, Merck Millipore), anti-human
Von Willebrand Factor (VWF DAKO A0082, Agilent Technologies), and anti-human CD68
(ab955, Abcam, Cambridge, United Kingdom) diluted in TBS-1%BSA-0.05% Tween (Tween
20, A4974,0250, AppliChem). Subsequently, samples were incubated for 1 h under agi-
tation at room temperature with secondary antibodies: goat anti-rat IgG AlexaFluor 680
(Invitrogen A21096, Thermo Fisher Scientific), goat anti-rat IgG AlexaFluor 568 (Invitrogen
A11077, Thermo Fisher Scientific), goat anti-rabbit IgG AlexaFluor 568 (Invitrogen A11036,
Thermo Fisher Scientific), donkey anti-mouse AF 568 (Invitrogen A10037, Thermo Fisher
Scientific), and goat anti-mouse IgG AlexaFluor 488 (Invitrogen A32766, Thermo Fisher
Scientific). All secondary antibodies were diluted in TBS-1%BSA-0.05%Tween. Slides
were washed, mounted with Prolong Diamond Antifade Mountant with DAPI (Invitrogen
P36962, Thermo Fisher Scientific) and imaged using a 20x objective on a Zeiss LSM980
confocal microscope (Zeiss, Oberkochen, Germany) and analyzed with Image J (version
2.3.0/1.53q).
2.11. Statistics
Data collection and analyses were performed with Excel (Microsoft, Redmond, WA,
USA) and Prism 9.0 (GraphPad). For survival data, Kaplan–Meier curves were plotted
and the Mantel–Cox log-rank test was used to determine significant differences between
groups. Data are presented either as single measurements or as group means
±
standard
deviations (s.d.).

Biomedicines 2024,12, 1927 6 of 21
3. Results
Here, we present the results from orthotopic cardiac pig-to-baboon xenotransplanta-
tion experiments with an immunosuppressive therapy based on a combination of CD40
and CD40L blockade. We compare this combination therapy to a previously published
study group treated only with the anti-CD40 Mab as co-stimulation blockade [
4
]. Hearts
from six genetically modified piglets, homozygous for
α
1,3-galactosyltransferase knock-
out (GGTA1-KO) and hemizygous transgenic for human CD46 (hCD46) as well as human
thrombomodulin (hTBM), were transplanted into male captive-bred baboons. In addi-
tion to an initial B cell depletion therapy with rituximab, all animals were continuously
dosed with a combination of an anti-CD40 IgG4 (initially 50 mg/kg body weight (bw),
lowered to
30 mg/kg bw)
and the anti-CD40L PAS-Fab (initially 20 mg/kg bw, lowered to
10 mg/kg bw) (Figure 1).
Biomedicines 2024, 12, x FOR PEER REVIEW 7 of 22
Figure 1. Dosing scheme (top) and survival (bottom) of the study group comprising six animals,
which received anti-CD40 IgG4/anti-CD40L PAS-Fab combination therapy (orange). The dosage of
the anti-CD40 Mab was reduced within 30 days from initially 50 to 30 mg/kg body weight (bw), and
the dosage of the anti-CD40L PAS-Fab was lowered within 60 days from 20 to 10 mg/kg bw. In
comparison with a previously reported [4] immunosuppressive regimen with the anti-CD40 Mab
alone (gray), there was no significant difference in survival (Log-rank (Mantel–Cox) test, n = 10, p =
0.0896). In the current study group, two technical failures were excluded from the survival analysis.
In the previously described group [4], two animals which were positive for PCMV/PRV were ex-
cluded. PCMV/PRV, porcine cytomegalovirus/porcine roseolovirus.
3.1. Recipient Survival Times
Mean survival in the current combination therapy group was 78 days, and maximum
survival was 170 days (Table 1). Hence, survival rates in the six xenotransplantation ex-
periments were not significantly different from the results of a previous group treated
with an immunosuppressive regimen based on monotherapy with the same anti-CD40
Mab alone [4] (p = 0.0896, Log-rank (Mantel–Cox) test, n = 10; Figure 1).
Table 1. Serum levels of heart and liver enzymes, platelet counts and prothrombin ratio at the end
of all six experiments.
Experiment #16956 #16935 #16950 #17353 #17012 #17020 Reference *
Bilirubin
[mg/dL] <0.1 <0.1 5.8 5.8 <0.1 <0.1 ≤1.2
AST [U/L] 27 64 1360 332 56 456 ≤49
PR [%] 88 128 13 41 141 60 70–130
CHE [kU
/
L] 112 13.4 1.2 1.9 1.6 8.6 4.6–11.5
Trop. T [ng/mL] <0.013 <0.013 10.30 4.85 0.043 0.373 ≤0.014
CK total [U/L] 47 79 18689 5826 116 501 ≤189
LDH [U/L] 361 757 13630 1028 729 2429 ≤249
Platelets [G/L] 498 498 367 42 118 386 150–300
Survival [days] 90 90 1 1 120 170
Figure 1. Dosing scheme (top) and survival (bottom) of the study group comprising six animals,
which received anti-CD40 IgG4/anti-CD40L PAS-Fab combination therapy (orange). The dosage
of the anti-CD40 Mab was reduced within 30 days from initially 50 to 30 mg/kg body weight (bw),
and the dosage of the anti-CD40L PAS-Fab was lowered within 60 days from 20 to 10 mg/kg bw. In
comparison with a previously reported [
4
] immunosuppressive regimen with the anti-CD40 Mab
alone (gray), there was no significant difference in survival (Log-rank (Mantel–Cox) test, n= 10,
p= 0.0896). In the current study group, two technical failures were excluded from the survival
analysis. In the previously described group [
4
], two animals which were positive for PCMV/PRV
were excluded. PCMV/PRV, porcine cytomegalovirus/porcine roseolovirus.
3.1. Recipient Survival Times
Mean survival in the current combination therapy group was 78 days, and maximum
survival was 170 days (Table 1). Hence, survival rates in the six xenotransplantation
experiments were not significantly different from the results of a previous group treated
with an immunosuppressive regimen based on monotherapy with the same anti-CD40
Mab alone [4] (p= 0.0896, Log-rank (Mantel–Cox) test, n= 10; Figure 1).

Biomedicines 2024,12, 1927 7 of 21
Table 1. Serum levels of heart and liver enzymes, platelet counts and prothrombin ratio at the end of
all six experiments.
Experiment #16956 #16935 #16950 #17353 #17012 #17020
Reference *
Bilirubin [mg/dL] <0.1 <0.1 5.8 5.8 <0.1 <0.1 ≤1.2
AST [U/L] 27 64 1360 332 56 456 ≤49
PR [%] 88 128 13 41 141 60 70–130
CHE [kU/L] 112 13.4 1.2 1.9 1.6 8.6 4.6–11.5
Trop. T [ng/mL] <0.013 <0.013 10.30 4.85 0.043 0.373 ≤0.014
CK total [U/L] 47 79 18689 5826 116 501 ≤189
LDH [U/L] 361 757 13630 1028 729 2429 ≤249
Platelets [G/L] 498 498 367 42 118 386 150–300
Survival [days] 90 90 1 1 120 170
Causes for euthanasia Study
endpoint
Study
endpoint
Technical
failure
(pulmonary
stenosis)
Technical
failure
(insufficient
perfusion)
Recalcitrant
pleural
effusions
Graft
failure
(humoral
rejection)
AST, aspartate aminotransferase; CHE, cholinesterase; Trop. T, troponin T; CK, creatine kinase; LDH, lactate
dehydrogenase; PR, prothrombin ratio. The two experiments #16950 and #17353 were excluded from further data
analysis as they had to be stopped within the first 24 h due to technical failures. *, Reference values as defined by
the Institute of Laboratory Medicine (University Hospital, LMU Munich, Munich, Germany).
Two of the six experiments (#16950 and #17353) had failed early due to technical
reasons. These two animals were excluded from further data analysis. Two more experi-
ments (#16956 and #16935) were deliberately terminated when the predetermined period
of
90 postoperative
days (set by the regulatory authorities) was reached with both recipi-
ents in excellent clinical condition. The final two experiments (#17012 and #17020) were
extended beyond 90 days (with permission from the regulatory authorities). Animal #17012
developed pleural effusions and was euthanized after 120 days. Animal #17020 appeared in
good clinical condition until day 165. Thereafter, the cardiac function deteriorated rapidly
due to acute humoral rejection, and the animal was euthanized after 170 days.
3.2. Organ Blood Parameters and Non-Gal-α(1,3)-Gal Xenoreactive Antibody Levels
Serum levels of the cardiac enzyme troponin T were high after surgery but dropped
subsequently to a normal range within the first postoperative week in all animals (Figure 2).
In the two experiments which were electively terminated after 90 days (#16956 and #16935),
troponin T levels were within a normal range throughout the study.
In the two animals that were extended beyond 90 days, troponin T levels gradually
increased towards the end of the experiment (Figure 2); this increase was especially pro-
nounced in baboon #17020 after day 160, thus indicating myocyte damage due to acute
humoral rejection. In baboon #17012, an early moderate increase in troponin T levels
coincided with the appearance of pleural effusion on day 30 (see below); however, at day
70 troponin T levels had returned to the normal range.
Other laboratory markers such as platelets, LDH, creatinine and bilirubin showed a
mostly inconspicuous time course in all experiments (Figure 3).
Only in baboon #17020, LDH levels started to increase after 100 days with a sharp peak
toward the end of the experiment, coinciding with the marked increase in troponin T level
(Figures 2and 3c). Notably, the analysis of IgM or IgG directed against non-Gal-
α
(1,3)-Gal
PAEC from GGTA1-KO,hCD46/hTBM transgenic animals showed no relevant increase in
all experiments (Figure 4).

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Causes for
euthanasia
Study
endpoint
Study
endpoint
Technical
failure
(pulmo-
nary ste-
nosis)
Technical
failure
(insuffi-
cient per-
fusion)
Recalci-
trant
pleural
effusions
Graft
failure
(humoral
rejection)
AST, aspartate aminotransferase; CHE, cholinesterase; Trop. T, troponin T; CK, creatine kinase;
LDH, lactate dehydrogenase; PR, prothrombin ratio. The two experiments #16950 and #17353 were
excluded from further data analysis as they had to be stopped within the first 24 h due to technical
failures. *, Reference values as defined by the Institute of Laboratory Medicine (University Hospital,
LMU Munich, Munich, Germany).
Two of the six experiments (#16950 and #17353) had failed early due to technical rea-
sons. These two animals were excluded from further data analysis. Two more experiments
(#16956 and #16935) were deliberately terminated when the predetermined period of 90
postoperative days (set by the regulatory authorities) was reached with both recipients in
excellent clinical condition. The final two experiments (#17012 and #17020) were extended
beyond 90 days (with permission from the regulatory authorities). Animal #17012 devel-
oped pleural effusions and was euthanized after 120 days. Animal #17020 appeared in
good clinical condition until day 165. Thereafter, the cardiac function deteriorated rapidly
due to acute humoral rejection, and the animal was euthanized after 170 days.
3.2. Organ Blood Parameters and Non-Gal-α(1,3)-Gal Xenoreactive Antibody Levels
Serum levels of the cardiac enzyme troponin T were high after surgery but dropped
subsequently to a normal range within the first postoperative week in all animals (Figure
2). In the two experiments which were electively terminated after 90 days (#16956 and
#16935), troponin T levels were within a normal range throughout the study.
Figure 2. Serum troponin T levels. Levels were high after surgery but subsequently dropped to a
normal range in animals #16956, #16935 and #17012. The strong increase in serum troponin T levels
Figure 2. Serum troponin T levels. Levels were high after surgery but subsequently dropped to a
normal range in animals #16956, #16935 and #17012. The strong increase in serum troponin T levels in
animal #17020 indicates myocardial damage due to graft rejection towards the end of the experiment.
Biomedicines 2024, 12, x FOR PEER REVIEW 9 of 22
in animal #17020 indicates myocardial damage due to graft rejection towards the end of the experi-
ment.
In the two animals that were extended beyond 90 days, troponin T levels gradually
increased towards the end of the experiment (Figure 2); this increase was especially pro-
nounced in baboon #17020 after day 160, thus indicating myocyte damage due to acute
humoral rejection. In baboon #17012, an early moderate increase in troponin T levels co-
incided with the appearance of pleural effusion on day 30 (see below); however, at day 70
troponin T levels had returned to the normal range.
Other laboratory markers such as platelets, LDH, creatinine and bilirubin showed a
mostly inconspicuous time course in all experiments (Figure 3).
(a) (b)
(c) (d)
Figure 3. Plasma bilirubin (a), creatinine (b) and LDH (c) levels and platelet counts (d), non-sugges-
tive of thrombotic microangiopathy. There were no signs of liver or kidney damage. The increase in
LDH in animal #17020 at the end of the experiment was caused by humoral rejection. LDH, lactate
dehydrogenase.
Only in baboon #17020, LDH levels started to increase after 100 days with a sharp
peak toward the end of the experiment, coinciding with the marked increase in troponin
T level (Figures 2 and 3c). Notably, the analysis of IgM or IgG directed against non-Gal-
α(1,3)-Gal PAEC from GGTA1-KO, hCD46/hTBM transgenic animals showed no relevant
increase in all experiments (Figure 4).
Mean fluoresence intensity
(a) (b)
Figure 3. Plasma bilirubin (a), creatinine (b) and LDH (c) levels and platelet counts (d), non-suggestive
of thrombotic microangiopathy. There were no signs of liver or kidney damage. The increase in
LDH in animal #17020 at the end of the experiment was caused by humoral rejection. LDH, lactate
dehydrogenase.

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in animal #17020 indicates myocardial damage due to graft rejection towards the end of the experi-
ment.
In the two animals that were extended beyond 90 days, troponin T levels gradually
increased towards the end of the experiment (Figure 2); this increase was especially pro-
nounced in baboon #17020 after day 160, thus indicating myocyte damage due to acute
humoral rejection. In baboon #17012, an early moderate increase in troponin T levels co-
incided with the appearance of pleural effusion on day 30 (see below); however, at day 70
troponin T levels had returned to the normal range.
Other laboratory markers such as platelets, LDH, creatinine and bilirubin showed a
mostly inconspicuous time course in all experiments (Figure 3).
(a) (b)
(c) (d)
Figure 3. Plasma bilirubin (a), creatinine (b) and LDH (c) levels and platelet counts (d), non-sugges-
tive of thrombotic microangiopathy. There were no signs of liver or kidney damage. The increase in
LDH in animal #17020 at the end of the experiment was caused by humoral rejection. LDH, lactate
dehydrogenase.
Only in baboon #17020, LDH levels started to increase after 100 days with a sharp
peak toward the end of the experiment, coinciding with the marked increase in troponin
T level (Figures 2 and 3c). Notably, the analysis of IgM or IgG directed against non-Gal-
α(1,3)-Gal PAEC from GGTA1-KO, hCD46/hTBM transgenic animals showed no relevant
increase in all experiments (Figure 4).
Mean fluoresence intensity
(a) (b)
Figure 4. Levels of non-Gal-
α
(1,3)-Gal xenoreactive IgM (a) and IgG (b) as measured by flow
cytometry on porcine aortic endothelial cells (PAEC) from GGTA1-KO,hCD46/hTBM transgenic
animals. The values from a previously investigated baboon that had rejected an intrathoracic
heterotopically transplanted pig heart served as positive control (black) and showed a strong increase
in both IgM and IgG, indicative of humoral rejection. Note: while animal #17020 revealed clinical
and histological signs of humoral rejection, there was no increase in xenoreactive IgM and IgG.
3.3. Levels of Inflammatory and Injury Markers
In contrast to the other animals, baboon #17012 developed recalcitrant pleural effusions
during the experiment (Figure 5a). Beginning around postoperative day 30, effusions
were initially mild and were controlled by occasional drainage. This first phase was
accompanied by a sharp increase in leukocytes and serum IL-6 levels but no relevant
increase in CRP (Figure 5b,c). The occurrence of pleural effusions also coincided with the
first rise in troponin T level (Figure 2), while other myocardial markers like NT-proBNP or
CK remained stable (Figure 5d).
After day 85, there was a sudden sharp rise in pleural effusions, needing more reg-
ular thoracocentesis. This second phase was also accompanied by increases in leukocyte
numbers, IL-6 (Figure 5b) and troponin T (Figure 2). In contrast to the first phase of pleu-
ral effusions around day 30, there were also relevant increases in NT-proBNP, CK, CRP
(Figure 5c,d) as well as several chemokines and pro-inflammatory cytokines, like MCP-1,
I-TAC, MIG, IL-6, IL-12, TNF-
α
and eotaxin (Figure 6). The other animals did not show any
relevant changes in chemokines and pro-inflammatory cytokines over the entire course of
the experiment, except for MCP-1 in baboons #16935, #17012 and, starting after day 110,
MCP-1, MIG, TNF-αand eotaxin in baboon #17020 (Figure 6a–g).
3.4. Necropsy and Histology
Histopathological analysis of the transplanted transgenic porcine hearts revealed
no evidence of antibody-mediated or cellular rejection in three of the four animals that
were under continued study (Figure 7a–e). However, the transplanted heart in baboon
#17020 showed focal capillaritis, interstitial edema, hemorrhages, acute myocardial necrosis
and distinct C4d positivity of capillaries, which are histological signs of severe antibody-
mediated rejection (pAMR3 according to the classification by the International Society for
Heart and Lung Transplantation, ISHLT) (Figure 7f–i).
Notably, all animals showed signs of fungal colonization in the lungs (Figure 7b,e,i;
not shown for baboon #16935). In addition, fungal emboli caused by yeast cells and hyphae
were sporadically found in lung arteries of baboon #16956; however, deep mycosis was not
detected (Figure 7b). While micromorphology did not allow for a specific diagnosis, size
and morphology were consistent with Candida species. The livers of baboons #16956, #16935
and #17020 showed hepatic steatosis to varying degrees. The diagnostic workup of the
other organs (e.g., kidneys, spleen, intestine) did not reveal any clinically
relevant findings.

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Figure 4. Levels of non-Gal-α(1,3)-Gal xenoreactive IgM (a) and IgG (b) as measured by flow cy-
tometry on porcine aortic endothelial cells (PAEC) from GGTA1-KO, hCD46/hTBM transgenic ani-
mals. The values from a previously investigated baboon that had rejected an intrathoracic hetero-
topically transplanted pig heart served as positive control (black) and showed a strong increase in
both IgM and IgG, indicative of humoral rejection. Note: while animal #17020 revealed clinical and
histological signs of humoral rejection, there was no increase in xenoreactive IgM and IgG.
3.3. Levels of Inflammatory and Injury Markers
In contrast to the other animals, baboon #17012 developed recalcitrant pleural effu-
sions during the experiment (Figure 5a). Beginning around postoperative day 30, effu-
sions were initially mild and were controlled by occasional drainage. This first phase was
accompanied by a sharp increase in leukocytes and serum IL-6 levels but no relevant in-
crease in CRP (Figure 5b,c). The occurrence of pleural effusions also coincided with the
first rise in troponin T level (Figure 2), while other myocardial markers like NT-proBNP
or CK remained stable (Figure 5d).
(a) (b)
(c) (d)
Figure 5. Cumulative pleural effusions in baboon #17012 (a) and serum levels of the inflammatory
markers leukocytes and IL-6 (b), CRP (c) as well as the myocardial markers CK and NT-proBNP (d).
The sharp rise in pleural effusions around postoperative day 85 was accompanied by a marked
increase in all inflammatory and myocardial markers (a–d and Figure 2). For comparison, at the
beginning of the pleural effusions around postoperative day 30 there was only an increase in IL-6,
leukocyte count (b) and also in troponin T levels (Figure 2).
After day 85, there was a sudden sharp rise in pleural effusions, needing more regu-
lar thoracocentesis. This second phase was also accompanied by increases in leukocyte
numbers, IL-6 (Figure 5b) and troponin T (Figure 2). In contrast to the first phase of pleural
effusions around day 30, there were also relevant increases in NT-proBNP, CK, CRP (Fig-
ure 5c,d) as well as several chemokines and pro-inflammatory cytokines, like MCP-1, I-
TAC, MIG, IL-6, IL-12, TNF-α and eotaxin (Figure 6). The other animals did not show any
Figure 5. Cumulative pleural effusions in baboon #17012 (a) and serum levels of the inflammatory
markers leukocytes and IL-6 (b), CRP (c) as well as the myocardial markers CK and NT-proBNP
(d). The sharp rise in pleural effusions around postoperative day 85 was accompanied by a marked
increase in all inflammatory and myocardial markers ((a–d) and Figure 2). For comparison, at the
beginning of the pleural effusions around postoperative day 30 there was only an increase in IL-6,
leukocyte count (b) and also in troponin T levels (Figure 2).

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relevant changes in chemokines and pro-inflammatory cytokines over the entire course of
the experiment, except for MCP-1 in baboons #16935, #17012 and, starting after day 110,
MCP-1, MIG, TNF-α and eotaxin in baboon #17020 (Figure 6a–g).
(a) (b)
MIG [pg/ml]
(c) (d)
(e) (f)
(g) (h)
Figure 6. Serum levels of different pro-inflammatory cytokines in all animals of the study group (a–
g). The marked increase around postoperative day 85 was only observed in baboon #17012 (ma-
genta), whereas #17020 showed elevated levels of some cytokines towards the end of the experi-
ment, when organ rejection occurred. The sharp increase in pleural effusions around postoperative
day 85 in baboon #17012 (h) was accompanied by a strong rise in several pro-inflammatory cytokine
levels (magenta).
Figure 6. Serum levels of different pro-inflammatory cytokines in all animals of the study group (a–g).
The marked increase around postoperative day 85 was only observed in baboon #17012 (magenta),
whereas #17020 showed elevated levels of some cytokines towards the end of the experiment,
when organ rejection occurred. The sharp increase in pleural effusions around postoperative day
85 in baboon #17012 (h) was accompanied by a strong rise in several pro-inflammatory cytokine
levels (magenta).

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3.4. Necropsy and Histology
Histopathological analysis of the transplanted transgenic porcine hearts revealed no
evidence of antibody-mediated or cellular rejection in three of the four animals that were
under continued study (Figure 7a–e). However, the transplanted heart in baboon #17020
showed focal capillaritis, interstitial edema, hemorrhages, acute myocardial necrosis and
distinct C4d positivity of capillaries, which are histological signs of severe antibody-me-
diated rejection (pAMR3 according to the classification by the International Society for
Heart and Lung Transplantation, ISHLT) (Figure 7f–i).
Notably, all animals showed signs of fungal colonization in the lungs (Figure 7b,e,i;
not shown for baboon #16935). In addition, fungal emboli caused by yeast cells and hy-
phae were sporadically found in lung arteries of baboon #16956; however, deep mycosis
was not detected (Figure 7b). While micromorphology did not allow for a specific diag-
nosis, size and morphology were consistent with Candida species. The livers of baboons
#16956, #16935 and #17020 showed hepatic steatosis to varying degrees. The diagnostic
workup of the other organs (e.g., kidneys, spleen, intestine) did not reveal any clinically
relevant findings.
Figure 7. Microscopic findings in post-mortem myocardial specimens. Histological analysis re-
vealed mild-to-marked interstitial edema (arrows) in myocardial specimens of baboons #16956 (a),
#16935 (c), #17012 (d) and #17020 (f,h). Fungal thrombi in pulmonal vessels (arrowhead) were de-
tected in all animals (#16956, b; #17012, e; #17020, i; not shown for #16935). Based on capillaritis
(arrowhead, f), endothelial swelling (arrowhead, h) and elongated C4d staining in capillaries (ar-
rows, g), baboon #17020 was diagnosed with severe antibody-mediated rejection (AMR3). a–d, f,h,i,
H&E staining; e, Grocott methenamine staining; g, C4d staining; scale bars = 100 µm.
Figure 7. Microscopic findings in post-mortem myocardial specimens. Histological analysis revealed
mild-to-marked interstitial edema (arrows) in myocardial specimens of baboons #16956 (a), #16935
(c), #17012 (d) and #17020 (f,h). Fungal thrombi in pulmonal vessels (arrowhead) were detected in all
animals (#16956, (b); #17012, (e); #17020, (i); not shown for #16935). Based on capillaritis (arrowhead,
(f)), endothelial swelling (arrowhead, (h)) and elongated C4d staining in capillaries (arrows, (g)),
baboon #17020 was diagnosed with severe antibody-mediated rejection (AMR3). (a–d,f,h,i), H&E
staining; (e), Grocott methenamine staining; (g), C4d staining; scale bars = 100 µm.
3.5. Analyses of Complement System, Coagulation System, Tissue Structure and Innate Immune
Cell Infiltration
In the two experiments deliberately terminated after 90 postoperative days (#16956
and #16935), immunofluorescence staining did not reveal notable antibody deposition,
complement activation, fibrin deposition or cellular infiltrates (macrophages) compared to a
non-transplanted, wild-type pig heart as the control (Figure 8a–d,f). The myocardial tissues
were overall healthy, with interspersed capillary distribution, well-organized polygonal
cardiomyocytes and discernible rounded nuclei (Figure 8e).
In the two experiments extended beyond 90 postoperative days (#17012 and #17020),
the immunofluorescence results showed a different pattern. A mild IgM antibody deposi-
tion and activation of the complement system (C4b/c, C3b/c and C5b, Figure 8a–c) were
clearly visible. Mild vascular and extravascular fibrin depots correlated with increased
structural alterations of the myocardial tissue as assessed by membrane staining with WGA
lectin (Figure 8d,e). Interestingly, there was also a massive infiltration of macrophages
(Figure 8f).
3.6. Virus Testing
The donor pigs as well as the baboon recipients were tested for PCMV/PRV using
real-time PCR, nested-PCR and PCMV/PRV glycoprotein B (gB)-based peptide ELISA
and Western blot assays. All tested organs of the baboons (skin, kidney, spleen, liver,

Biomedicines 2024,12, 1927 13 of 21
lung, peripheral blood mononuclear cells) as well as the explanted transgenic pig heart
were negative for PCMV/PRV as well as PCV3 and PLHV-3. Preventing the transmission
of PCMV/PRV, PLHV-3 and PCV3 to the baboon recipients significantly contributed to
their survival because pig hearts infected with PCMV/PRV typically survive less than
30 days [42].
Since the antiviral substances ganciclovir and valganciclovir are effective
against human cytomegalovirus (HCMV) but not against PCMV/PRV, which is a rose-
olovirus [
43
–
45
], they should not be administered for the prevention of a PCMV/PRV
infection. Of note, PCMV/PRV was also transmitted to the first recipient of a pig heart in
Baltimore [8,9] and may have contributed to the early death of this patient.
Biomedicines 2024, 12, x FOR PEER REVIEW 14 of 22
Figure 8. Immunofluorescence staining of post-mortem myocardial specimens. Antibody deposi-
tion (IgM) (a), complement deposition (C3b/c, C4c, C5b) (b,c), fibrin deposition (Fgn) (d), cardiomy-
ocyte structure (WGA, a lectin staining N-acetyl-D-glucosamine and sialic acid on the cell mem-
brane) (e) and macrophage infiltration (CD68) (f) were analyzed in all donor organs. Scale bar, 100
µm. n = 4 GGTA1-KO, hCD46/hTBM transgenic pigs; n = 1 wild-type pig (control).
4. Discussion
There are several studies demonstrating the therapeutic potential of agents interfer-
ing with the CD40/CD40L immune checkpoint, but it is still unclear whether a single Mab
or a combination of two or more Mabs may be better at preventing xenograft rejection
[10].
4.1. Rationale for a Combination Therapy With Anti-CD40 and Anti-CD40L Antibodies
When using an anti-CD40 Mab for co-stimulation blockade in preclinical non-human
primate cardiac xenotransplantation models, a dosage of 50 mg/kg bw seemed appropri-
ate for long-term xenograft survival [14,15,21]. When lower dosages were given [21], or
when the dosage was tapered out over the course of the experiment [14,15], xenografts
were ultimately rejected. However, the high dosage of 50 mg/kg bw might not be feasible
in future human applications (amounting to several grams of antibody to be administered
per dose). As described above, a combination of the anti-CD40 and anti-CD40L antibodies
may have synergistic effects in vivo, which should allow dose reduction for each agent.
To date, a combination of anti-CD40 and anti-CD40L Mabs has not been published, and
we present the first description of such an approach.
Thrombotic side effects were described for the first-generation anti-CD40L Mab
[16,17] and, consequently, the clinical development of ruplizumab (hu5c8) was halted. As
the Fc-part of the Mab was found to be the cause of this detrimental effect [17,46], this
portion had been removed in the anti-CD40L PAS-Fab which was used in the current
Figure 8. Immunofluorescence staining of post-mortem myocardial specimens. Antibody deposition
(IgM) (a), complement deposition (C3b/c, C4c, C5b) (b,c), fibrin deposition (Fgn) (d), cardiomyocyte
structure (WGA, a lectin staining N-acetyl-D-glucosamine and sialic acid on the cell membrane)
(e) and
macrophage infiltration (CD68) (f) were analyzed in all donor organs. Scale bar, 100
µ
m. n= 4
GGTA1-KO,hCD46/hTBM transgenic pigs; n= 1 wild-type pig (control).
4. Discussion
There are several studies demonstrating the therapeutic potential of agents interfering
with the CD40/CD40L immune checkpoint, but it is still unclear whether a single Mab or a
combination of two or more Mabs may be better at preventing xenograft rejection [10].
4.1. Rationale for a Combination Therapy with Anti-CD40 and Anti-CD40L Antibodies
When using an anti-CD40 Mab for co-stimulation blockade in preclinical non-human
primate cardiac xenotransplantation models, a dosage of 50 mg/kg bw seemed appropriate
for long-term xenograft survival [
14
,
15
,
21
]. When lower dosages were given [
21
], or when
the dosage was tapered out over the course of the experiment [
14
,
15
], xenografts were
ultimately rejected. However, the high dosage of 50 mg/kg bw might not be feasible in

Biomedicines 2024,12, 1927 14 of 21
future human applications (amounting to several grams of antibody to be administered per
dose). As described above, a combination of the anti-CD40 and anti-CD40L antibodies may
have synergistic effects
in vivo
, which should allow dose reduction for each agent. To date,
a combination of anti-CD40 and anti-CD40L Mabs has not been published, and we present
the first description of such an approach.
Thrombotic side effects were described for the first-generation anti-CD40L Mab [
16
,
17
]
and, consequently, the clinical development of ruplizumab (hu5c8) was halted. As the
Fc-part of the Mab was found to be the cause of this detrimental effect [
17
,
46
], this portion
had been removed in the anti-CD40L PAS-Fab which was used in the current study. The
resulting shorter plasma half-life of the monovalent antibody fragment—which also offers
the functional benefit that it cannot cross-link its antigen—was extended using PASylation
technology [
33
]. This novel immunosuppressive agent was successfully applied as single
therapy in xenotransplantation studies before [3].
4.2. Outcome of a Combination Therapy of Anti-CD40 IgG4 and Anti-CD40L PAS-Fab
We initially administered both antibody agents at high dosages, which were decreased
after 30 or 60 days, respectively. The aim of this procedure was to reduce the amount of
drug substance administered to the patient in a potential clinical setting. The results of this
animal study proved that, in principle, a combination therapy of anti-CD40 and anti-CD40L
is effective and well tolerated (except for the fungal infections, see below). There was no
significant difference in survival between the current combination therapy group and a
previous study group using only the anti-CD40 Mab [
4
]. Three of four animals showed
rejection-free survival for up to 120 days. No large-vessel thrombogenic complications
were seen in any animal of the study group. This needs to be emphasized, as thrombotic
side effects were described for a first-generation anti-CD40L Mab [
16
,
17
]. Nevertheless, the
fourth animal succumbed to acute humoral rejection after 170 postoperative days.
During the first 90 postoperative days, there was no relevant difference in clinical
appearance, vital signs or laboratory analyses among the four animals investigated here
and compared to previous study groups using either the anti-CD40 IgG4 or anti-CD40L
PAS-Fab [
3
,
4
]. Only the two present experiments extending beyond 90 days (#17012 and
#17020), i.e., 60/30 days after decreasing antibody doses, exhibited a conspicuous course, as
demonstrated, for example, by increasing serum levels of LDH, AST (#17020) and troponin
T (#17012 and #17020, see below). This is consistent with previous data, when serum LDH
of >600 U/L and AST of >300 U/L were associated with the development of acute humoral
rejection after cardiac xenotransplantation [47].
Interestingly, animal #17020, which suffered from severe humoral rejection, showed
no relevant increase in non-Gal-
α
(1,3)-Gal xenoreactive IgM or IgG in the plasma. Of
note, the immunochemical assay applied [
41
] only served to detect non-donor-specific
circulating antibodies directed against surface antigens of PAEC from the GGTA1-KO,
hCD46/hTBM transgenic donor pigs, but not those reactive with intracellular or secreted
porcine proteins. This constellation was also observed in heterotopic abdominal cardiac
xenotransplantation experiments when non-Gal-
α
(1,3)-Gal xenoreactive antibodies were
not always detected at the time of graft or recipient demise [
14
]. Nevertheless, the authors
of that study hypothesized that there was incomplete control of anti-pig immunity in these
animals [14].
In contrast, immunofluorescence analyses of the myocardial tissue from animal #17020
revealed the deposition of IgM antibodies and of complement (C4b/c, C3b/c, and C5b).
There are two possible explanations for these apparently contradictory findings: (i) The anti-
non-Gal-
α
(1,3)-Gal xenoreactive antibodies produced by the baboon may almost entirely
be absorbed by the porcine cardiac xenograft and are therefore difficult to be detected in
plasma. (ii) The antibodies could be specific for the SLA-II antigen of the donor and would
not be detected by FACS with GGTA1-KO,hCD46/hTBM transgenic PAEC, as SLA-II is only
expressed on activated porcine endothelium. Further experiments are therefore needed

Biomedicines 2024,12, 1927 15 of 21
to identify the targets of the putative anti-donor antibodies, and the currently used assay
needs to be adapted or complemented with specific detection of anti-SLA-II.
Another interesting finding was a correlation between the courses of increased MCP-1
levels and macrophage infiltration in three of the four animals, i.e., #16935, #17012 and
#17020. The involvement of macrophages is a prominent feature of the innate immune
response and has been described in xenograft rejection before [
48
]. MCP-1 (monocyte
chemoattractant protein-1) is one of the key chemokines that regulate migration and
infiltration of monocytes/macrophages into inflammatory tissue sites and, thus, elevated
levels of MCP-1 may play a crucial role in graft survival [
49
]. While prevention of graft
rejection mediated by innate immune cells was not in the focus of the present study, this
needs to be investigated more closely in future experiments.
4.3. Myocardial Integrity of the Xenografts
As mentioned above, a conspicuous course of the myocardial integrity marker tro-
ponin T was only present in the two experiments extended beyond 90 days, #17012 and
#17020. The increase in troponin T after day 100, i.e., 70/40 days after decreasing the
antibody doses, was nearly identical in both animals and correlated with tissue damage
detectable as mild vascular and extravascular fibrin depots with increased alteration of the
myocardial structure. We assume that these changes should be considered in the context of
a multifactorial genesis.
In part, the increase in troponin T levels after day 100 could have been caused by an
insufficient immunosuppression as a result of reducing the doses of both anti-CD40 Mab
and anti-CD40L PAS-Fab. In a published heterotopic abdominal model, the reduction in
the anti-CD40 Mab monotherapy dose resulted in the recrudescence of anti-pig antibody
and graft failure [
14
]. In our current study group, we cannot clarify whether decreasing
the dose of either the anti-CD40 Mab, the anti-CD40L PAS-Fab, or of both agents, may
have caused the pathological observations. However, the assumption of an insufficient
immunosuppression is supported by the fact that necropsy of baboon #17020 showed signs
of severe humoral rejection.
The changes in troponin T levels could also have been caused by disproportionate
myocardial growth or xenogeneic hypertrophic obstructive cardiomyopathy [
50
]. This
phenomenon was strongly attenuated, but not totally stopped, by our regimen of growth
inhibition, including temsirolimus, antihypertensive treatment and early weaning from
cortisone [
3
,
50
]. Furthermore, fungal infections (see below) are also known to cause
elevation of troponin T levels [51,52].
4.4. Pleural Effusions in Animal #17012
The development of pleural effusions in animal #17012 was seen in two phases, which
both came along with an increase in leukocyte numbers, IL-6 and troponin T levels. During
the second phase, there was an additional increase in NT-proBNP, CK, CRP as well as
several pro-inflammatory cytokines. The rise in leukocytes, CRP and IL-6 indicates the
occurrence of inflammatory processes coinciding with the development of the pleural
effusions. Furthermore, the massive complement deposition observed in baboon #17012
leads us to the assumption that complement activation may also have contributed to the
pleural effusions. Besides this, some myocardial damage seems to have occurred, which is
indicated by the concomitant increase in troponin T, NT-proBNP and CK.
To our knowledge, there is no long-term data on pro-inflammatory cytokines after
orthotopic cardiac xenotransplantation available so far. In cardiac allotransplantation re-
cipients, levels of chemokines and pro-inflammatory cytokines increase in the months
following transplantation, possibly mediating endothelial damaging [
53
] and the recruit-
ment of more xenoreactive immune cells into the graft [
54
]. Chemokines such as I-TAC
or MIG are modulators of intragraft inflammation by recruiting CXCR3
+
T-cells [
55
,
56
].
Furthermore, it is known that pro-inflammatory cytokines such as TNF
α
may affect en-
dothelial function [
57
,
58
]. In lung allotransplantation, pro-inflammatory cytokines seem

Biomedicines 2024,12, 1927 16 of 21
to be involved in transplant outcome [
59
,
60
] and are associated with pro-inflammatory
responses leading to primary graft dysfunction [
61
]. Moreover, previous studies in murine
models of allogeneic cardiac transplantation indicated that antibodies blocking cytokines
such as IL-12 support graft survival by preventing Th1 and Th17 responses [62].
Taking these findings together, there might have been several factors responsible for
the recalcitrant effusions in baboon #17012: infectious/inflammatory processes, myocardial
damage, endothelial dysfunction and insufficient immunosuppression. Our analysis is
the first of its kind and therefore provides important clues regarding pleural effusions
as a challenge after pig-to-baboon cardiac xenotransplantation. Further studies, espe-
cially with a larger number of animals, should help to gain a better understanding of
the incidences, causes and consequences of pleural effusions after pig-to-baboon cardiac
xenotransplantation.
4.5. Fungal Infections
Necropsy data of all four animals of the current study group showed fungal infections,
which was unexpected, especially as the animals did not show any corresponding clinical
signs throughout the experiments. Fungal infections of such significant extent were not
observed in other xenografted animal groups studied in our laboratory (data partially
not published) [
3
,
4
,
63
,
64
] or in experiments reported by other research groups [
5
,
14
,
65
].
Therefore, antifungal prophylaxis was not part of the therapeutic regimen in the current
study group. For comparison, in cardiac, lung, liver or pancreas allotransplantation,
patients have a relevant incidence of 1% to 40% of fungal infections [
66
–
68
]. In clinical
practice, this can be anticipated by antifungal prophylaxis, which is well established in
human allotransplantation [
68
–
70
] and may also be an option for future preclinical and
clinical xenotransplantation.
Beyond the anti-CD40 IgG4/anti-CD40L PAS-Fab combination therapy, all animals
received mycophenolate mofetil as a generally immunosuppressive drug, which is known
to increase the risk of fungal infections [
71
–
73
]. However, since the animals in the afore-
mentioned studies [
3
–
5
,
14
,
63
–
65
] had also received this drug, we assume that the fungal
infections were more likely caused by the antibody combination therapy. CD40/CD40L
signaling is known to contribute to the adaptive Th1 immune response against fungi, such
as Candida albicans, and life-threatening fungal infections were reported in patients deficient
in CD40L or CD40 [
74
,
75
]. Indeed, the higher susceptibility for infectious side effects by all
treated animals may provide an indication that the suppression of this particular immune
checkpoint was overly strong in our study.
Interestingly, fungal infections were only diagnosed through necropsy and histological
examination after the experiments had been stopped. During the experiments, there were
no specific signs of fungal infections, such as in blood culture examinations or other samples.
Consequently, determining the precise onset of these infections is challenging. We assume
that they likely originated during the period prior to reducing the doses of the combination
therapy, resulting in an overly strong cellular and humoral immunosuppressive effect. The
pathologies in animals #17012 and #17020 (i.e., pleural effusions, increase in inflammatory
markers, humoral rejection, see above) clearly manifested after decreasing the anti-CD40
IgG4 and anti-CD40L PAS-Fab doses, and there were no comparable events in the animals
#16956 and #16935, which were deliberately terminated after 90 postoperative days. It is
tempting to speculate that the ’ideal’ dosage for the combination therapy of anti-CD40 IgG4
and anti-CD40L PAS-Fab may fall within the range between the high (promoting fungal
infections in all animals) and low dosages (permitting humoral rejection and recalcitrant
pleural effusions) administered in the current study group. This aspect should be addressed
in future studies on a combination therapy of antibodies blocking both CD40 and CD40L.
Besides fungal infections, xenotransplantation with corresponding anti-CD40-based im-
munosuppression can be associated with the activation of latent herpes viruses, including
HCMV, as well as external infections such as Pneumocystis pneumonia [
76
,
77
]. However, it
is quite feasible to diagnose and treat these infections.

Biomedicines 2024,12, 1927 17 of 21
5. Conclusions
Our experience with the first combination therapy of anti-CD40 and anti-CD40L
antibodies proved that such therapy is effective and generally well-tolerated. However,
there were also side effects/adverse events like humoral rejection, susceptibility to fungal
infections and recalcitrant effusions. Given the critical role of dosage in the balance between
infection and organ rejection, further trials and broader immunologic monitoring are
necessary to obtain a deeper understanding of the potential benefits of an anti-CD40 and
anti-CD40L co-stimulation blockade in preclinical cardiac xenotransplantation.
Author Contributions: Conceptualization, M.B., J.-M.A., B.R., R.R., U.B., M.G., A.S. and M.L.
(Matthias Längin); Data curation, M.B., J.-M.A. and M.L. (Matthias Längin); Formal analysis, M.B.,
J.-M.A. and M.L. (Matthias Längin); Funding acquisition, J.-M.A., B.R., E.W., P.B. and M.L. (Matthias
Längin); Investigation, M.B., J.-M.A., J.M.B., C.F., A.M., V.Z., R.R., U.B., M.G., A.S. and M.L. (Matthias
Längin); Methodology, M.B., J.-M.A. and M.L. (Matthias Längin); Project administration, J.-M.A.,
B.R., C.H., E.W., M.S., P.B. and M.L. (Matthias Längin); Resources, E.N., J.R., M.M., I.B., M.L. (Maria
Leuschen), F.W., S.M., R.E., S.S., A.P., A.L., B.K., E.K., N.K., J.D., A.W.G., R.R.T., J.M.B., C.F., A.M.,
V.Z., N.S.-A., A.D., R.R., S.L., C.W., C.H., D.A., E.W., M.S. and P.B.; Software, M.B., J.-M.A. and
M.L. (Matthias Längin); Supervision, J.-M.A., B.R., A.S. and M.L. (Matthias Längin); Validation, M.B.
and M.L. (Matthias Längin); Visualization, M.B., V.Z., R.R., S.L., C.W. and M.L. (Matthias Längin);
Writing—original draft, M.B.; Writing—review and editing, J.-M.A., J.D., A.W.G., R.R.T., C.F., A.M.,
V.Z., R.R., S.L., C.W., E.W., U.B., M.G., A.S. and M.L. (Matthias Längin). All authors have read and
agreed to the published version of the manuscript.
Funding: This research was funded by the German Research Foundation (Deutsche Forschungsge-
meinschaft, DFG) TRR 127 and, in part, by the Swiss National Science Foundation (CR-SII5_198577)
and the Leducq Foundation (23CVD01).
Institutional Review Board Statement: The animal study protocol was approved by the Institutional
Review Board (or Ethics Committee) of the Government of Upper Bavaria (Regierung von Oberbayern,
protocol codes ROB-55.2-2532.Vet_02-14-184, date of approval: February 09, 2015 and ROB-55.2-
2532.Vet_02-19-158, date of approval: 3 March 2020).
Data Availability Statement: The raw data supporting the conclusions of this article will be made
available by the authors on request.
Acknowledgments: The authors thank the German Primate Center and the Walter Brendel Centre of
Experimental Medicine for support and provision of facilities, especially D. Merkus, M. Shakarami
and all animal care takers. They acknowledge K. Reiman for providing the anti-CD40 monoclonal
antibody for the experiments.
Conflicts of Interest: Jan-Michael Abicht, Bruno Reichart, Eckhard Wolf, Paolo Brenner, Arne Skerra
and Matthias Längin are founders of XTransplant GmbH. David Ayares is chief executive officer and
chief scientific officer of Revivicor, Inc. Michaela Gebauer is an employee, Uli Binder and Arne Skerra
are shareholders of XL-protein GmbH. The funders had no role in the design of the study; in the
collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to
publish the results. The other authors declare no conflicts of interest.
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