CN107474143B

CN107474143B - (Anti-CD40mAb) -CTLA4 fusion protein and uses thereof

Info

Publication number: CN107474143B
Application number: CN201710810984.3A
Authority: CN
Inventors: 张军方
Original assignee: Shenzhen Yinnuosai Biology Technology Co ltd
Current assignee: Shenzhen Yinnuosai Biology Technology Co ltd
Priority date: 2017-09-13
Filing date: 2017-09-13
Publication date: 2020-08-11
Anticipated expiration: 2037-09-13
Also published as: CN107474143A

Abstract

The invention discloses an anti-immune rejection bifunctional molecule. According to the invention, the Anti-human CD40 antibody and CTLA4 protein are subjected to fusion expression to prepare the (Anti-CD40mAb) -CTLA4 fusion protein, and the fusion protein can simultaneously block the pathways of CD40-CD154 and CD28-B7, and has the functions of simultaneously blocking the activation of T, B cells and inducing immune tolerance. The invention is useful for reducing or treating graft rejection and graft-versus-host disease, as well as for treating autoimmune disease.

Description

(Anti-CD40mAb) -CTLA4 fusion protein and uses thereof

Technical Field

The invention relates to the technical field of antibody drugs, in particular to a (Anti-CD40mAb) -CTLA4 fusion protein prepared by fusion expression of an Anti-human CD40 antibody and CTLA4 protein and a preparation method thereof. The invention is useful for reducing or treating graft rejection and graft-versus-host disease, as well as for treating autoimmune disease.

Background

The invention relates to a method for expressing an anti-CD40 antibody and CTLA4 protein in a fusion mode and application of the anti-CD40 antibody and the CTLA4 protein in the fusion mode, for example, immune rejection in organ transplantation is reduced, and immune tolerance is induced to treat autoimmune diseases.

Reducing immune rejection is a problem that is commonly faced in the treatment of organ transplantation and autoimmune diseases. Autoimmune diseases affect 5% of the world population, and serious autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus can cause disability and even death. Organ transplantation as an important means of treating organ failure, 150 million patients need organ transplantation every year in China alone, however, the long-term survival of transplanted organs still faces a significant challenge so far, and host immune rejection results in graft function loss. Currently clinically used non-specific immunosuppressive drugs, although capable of suppressing rejection in the short term, have inadequate long-term results and the use of these agents greatly increases the risk of cardiovascular disease, infection and malignancy.

The interaction between T cells and B cells is critical in immune responses, where many molecules located on the surface of T cells and B cells are expressed in increased amounts. T cell activation relies on two signals, a first signal mediated by MHC-antigen peptides on the surface of Antigen Presenting Cells (APC) which are recognized by the T Cell Receptor (TCR) and bound thereto, and a second signal provided by the binding of CD28 on the surface of T cells to B7 molecules on the surface of APC, which are activated only in the presence of both signals, and which are clonally anergic or unresponsive in the absence of the assistance of the second signal. Cytotoxic T lymphocyte associated antigen-4 (CTLA-4) and CD28 have very similar relationship in gene structure, chromosome positioning and sequence homology, are all B7 receptors, and are mainly expressed on the surface of activated T cells. However, the functions of CTLA-4 and CD28 are opposite, and CTLA-4 combined with B7 inhibits T cell activation and plays a negative regulation role. The binding affinity of CTLA-4 and B7 is 10-20 times higher than that of CD28 and B7, and the binding of CD28 and B7 can be blocked by only a small amount of CTLA-4, so that the activation of T cells is inhibited. CTLA4-Ig is a fusion protein of extracellular domain of CTLA4 molecule produced by gene recombination and immunoglobulin IgG constant region, and has high affinity with B7 molecule and can compete with CD28 to bind to B7 family molecules CD80(B7-1) and CD86(B7-2) on APC, and block the signal path between CD28 and B7.

CD40-CD154 is regarded as an important co-stimulatory signaling pathway, mediates important processes such as activation, proliferation and differentiation of B cells and activation of T cells, and blocking the pathway is known to significantly reduce immune rejection. Early antibodies against CD154 caused severe thrombotic responses in clinical trials and were terminated, and research turned to the development of antibodies targeting CD 40. A large number of in vivo experiments prove that the CD 40-directed antibody can obviously reduce immunological rejection, does not cause toxic and side effects such as thrombus and the like, and has good development prospect. CD40 is expressed on the surface of B cells, macrophages and dendritic cells and is critical for B cell activation, immunoglobulin class switching, and ideally CD40 antibodies can inhibit B cell activation without activating B cells or causing B cell depletion.

Disclosure of Invention

CD28-B7 and CD40-CD154 are the two most important co-stimulatory signaling pathways in vivo that mediate the activation of T cells and B cells, respectively. CTLA4-Ig was able to specifically block the CD28-B7 pathway but not the CD40-CD154 pathway, and anti-CD40 antibodies were able to specifically block the CD40-CD154 pathway but not the CD28-B7 pathway. The research utilizes a genetic engineering method, and prepares a bifunctional molecule (Anti-CD40mAb) -CTLA4 fusion protein) which can simultaneously and efficiently block two signal paths of CD28-B7 and CD40-CD154 by fusing and expressing an Anti-CD40 antibody and CTLA4, thereby realizing' Erie. In vitro experiments, (Anti-CD40mAb) -CTLA4 was able to not only transform, proliferate, activate T cells but also inhibit B cell activation, in vivo experiments in macaques, (Anti-CD40mAb) -CTLA4 significantly blocked T cell-dependent Anti-KLH antibody responses and significantly prolonged survival of islet xenografts in a macaque model of xenograft islet transplantation.

According to the first aspect of the invention, the invention provides a bifunctional molecule for simultaneously and efficiently blocking two signal paths of CD28-B7 and CD40-CD154, wherein (Anti-CD40mAb) and CTLA4 fusion protein can not only transform, proliferate and activate T cells, but also inhibit the activation of B cells, and the molecule can significantly block T cell-dependent Anti-KLH antibody reaction, thereby significantly prolonging the survival of islet allografts in a macaque model of xenoislet transplantation.

Further, the above molecules include anti-human CD40 antibody and CTLA4 molecule.

Further, CTLA4 in the above molecule is linked to the heavy chain C-terminus of anti-human CD40 antibody.

Further, CTLA4 in the above molecule is linked to anti-human CD40 antibody directly or mediated by a linking sequence.

Further, the sequence of the above molecule is selected from SEQ ID NO: 1-8.

According to a second aspect of the invention, there is provided a method of fusing CTLA4 with an anti-human CD40 antibody.

According to a third aspect of the invention, there is provided an anti-immune rejection medicament. The invention is useful for reducing or treating graft rejection and graft-versus-host disease, as well as for treating autoimmune disease.

Further, the above drug is a fusion molecule of CTLA4 and an anti-human CD40 antibody.

Further, the above-mentioned drug is an anti-immune rejection drug.

Further, the use of the above medicament for reducing or treating transplant rejection and graft versus host disease, as well as for treating autoimmune diseases.

Drawings

FIG. 1 is a graph showing the reactivity of Anti-hCD40 monoclonal antibody with a native antigen in one embodiment of the present invention;

FIG. 2 is a graph of the pattern of activation of B cells blocked by the CD40 antibody in one embodiment of the invention;

FIG. 3 is a summary of analysis of Anti-hCD40 mab inhibition of B cell activation in one embodiment of the present invention;

FIG. 4 shows the expression scheme of the CD40 antibody fused with CTL4A in one embodiment of the present invention. The CH3 terminal of the CD40 antibody is connected with the extracellular segment of CTLA4 through a linker to prepare a bifunctional fusion protein;

FIG. 5 is a diagram showing the expression pattern of the CD40 antibody fused with CTL4A in one embodiment of the present invention;

FIG. 6 shows that (Anti-CD40mAb) -CTLA4 significantly inhibited T cell transformation in one embodiment of the invention;

FIG. 7 shows that (Anti-CD40mAb) -CTLA4 significantly inhibited MLR in one embodiment of the invention;

FIG. 8 shows that (Anti-CD40mAb) -CTLA4 inhibits the expression of the human peripheral blood T cell surface molecule CD25 in one embodiment of the present invention;

FIG. 9 shows that (Anti-CD40mAb) -CTLA4 inhibits human peripheral blood B cell activation in one embodiment of the invention;

FIG. 10 is a graph showing that (Anti-CD40mAb) -CTLA4 significantly blocks T cell-dependent Anti-KLH antibody responses in one embodiment of the present invention;

table one is the islet survival time in a cynomolgus monkey model for xenogenic islet transplantation in one embodiment of the present invention.

Detailed Description

The present invention will be described in further detail with reference to the following detailed description and accompanying drawings.

Example 1: preparation and characterization of CD40 antibody

Mice (BALB/C) were immunized multiple times with the extracellular domain of human CD40 protein (Met1-Arg193), Freund's adjuvant, and incomplete Freund's adjuvant. Splenocytes from immunized mice were fused with the mouse myeloma cell line SP2/0 and heterozygotes were selected using standard hybridoma techniques. Human CD40 protein is coated on an ELISA plate, positive cell strains are screened, and three times of cloning are carried out, so that the anti-human CD40 monoclonal antibody is finally obtained. The obtained monoclonal antibody is tested for reactivity with natural CD40 expressed on B cells in human blood by flow cytometry, and monoclonal antibodies capable of blocking B cell activation are screened in vitro as final antibodies.

Example 2: reactivity identification of Anti-hCD40 monoclonal antibody and natural CD40

Raji cells are a B-cell lymphoma cell line with high expression of CD40 molecule on its surface. To test the reactivity of the monoclonal antibody to hCD40 against the native antigen, flow cytometry analysis was used for identification. Respectively incubating the 38 antibody and the Raji cells, adding a secondary antibody marked with fluorescence, and finally detecting the fluorescence intensity by using a flow cytometer, wherein the fluorescence intensity can indicate the reactivity of the monoclonal antibody and a cell surface antigen. The reaction principle is shown in FIG. 1A, and the flow-type results are shown in FIG. 1B. From the results shown in the figure, most antibodies were highly reactive with the natural antigen, and only three antibodies were weakly reactive.

Example 3: analysis of Anti-hCD40 monoclonal antibody blocking B activation effect

CD40/CD154 is a positive regulatory signal that plays an important role in humans, and by inhibiting this signaling pathway, immune responses can be reduced. CD40 is used as a co-stimulation factor and is mainly expressed on the cell surface of B cells and other APC, and the combination of CD40 and CD154 provides a co-stimulation signal for the B cells, so that the B cells are fully activated, and the expression of CD80/CD86 is up-regulated. The subject group established a HEK-293 cell line stably expressing CD154, cultured for 6 days with isolated healthy human Peripheral Blood Mononuclear Cells (PBMC), and examined the expression of CD80/CD86 on the B cell surface using flow cytometry, as shown in FIG. 2. The results of the 38 strains of CD40 monoclonal antibodies are shown in FIG. 3, and most antibodies can obviously reduce the expression of CD80/CD86 and block the activation of B cells.

Example 4: humanization engineering of antibodies

The variable region sequences of the antibodies were PCR cloned using methods known in the art, and the PCR products were assembled with commercially available vectors and the sequences of the antibodies were obtained using standard sequencing. The heavy chain variable region and the light chain variable region of the antibody were cloned into expression vectors containing the human IgG4 heavy chain and human kappa light chain constant regions, respectively. The 3D conformation of the antibody is simulated by a computer, the murine part in the variable region sequence of the chimeric antibody is gradually replaced by the human sequence, and meanwhile, the affinity of the antibody is not remarkably reduced. After multiple modification expression identification, the humanized CD40 antibody with similar affinity to the original mouse monoclonal antibody is finally obtained.

Example 5: construction of (Anti-CD40mAb) -CTLA4 fusion protein

The research firstly screens out a CD40 antibody which can block a CD40-CD154 pathway with high efficiency, and the blocking effect of the CD40 antibody is superior to that of the reported CD40 antibody. By using a genetic engineering method, the CH3 terminal of a CD40 antibody is connected with an extracellular segment of CTLA4 through a linker to prepare a bifunctional fusion protein, as shown in the following figure 4, and a schematic diagram is shown in figure 5.

The research utilizes a genetic engineering method to prepare a bifunctional molecule which can simultaneously and efficiently block two signal paths of CD28-B7 and CD40-CD154 by fusing and expressing a CD40 antibody and CTLA4, thereby realizing' Erie.

Example 6: (Anti-CD40mAb) -CTLA4 significantly inhibited T cell transformation

T lymphocytes cultured in vitro can be transformed into lymphoblasts and undergo mitosis when stimulated with mitogens (e.g., PHA, ConA, etc.) and antigens, and this transformation reaction reflects the immune function status of body cells to some extent and can be used as an index for measuring cellular immune response. We examined the effect of BSA (control), CTLA4-Ig, Anti-CD40mAb, (Anti-CD40mAb) -CTLA4 on T cell transformation using PHA-stimulated human peripheral blood lymphocyte transformation as an index by the MTT method. As a result, CTLA4-Ig and (Anti-CD40mAb) -CTLA4 were found to significantly inhibit lymphocyte transformation, while Anti-CD40mAb was found not, as shown in FIG. 6.

Example 7: (Anti-CD40mAb) -CTLA4 significantly inhibited MLR

We stimulated peripheral blood T cells with allogeneic lymphocytes and observed the effect of (Anti-CD40mAb) -CTLA4 on this activation process. Incorporation of radioisotopes on the basis of cells³The strength of H-TdR, and the activation of T cells is judged. The result shows that (Anti-CD40mAb) -CTLA4 inhibits the activation of allogenic lymphocytes on peripheral blood T lymphocytes, and the average inhibition rate reaches 88.9%; the same amount of CTLA4-Ig inhibited 87%, while Anti-CD40mAb inhibited only 8.4%. Statistical analysis showed that the (Anti-CD40mAb) -CTLA4 group was significantly different from the CTLA4-Ig group compared to the control group (BSA), significantly inhibiting MLR while Anti-CD40mAb did not function as shown in FIG. 7.

Example 8: (Anti-CD40mAb) -CTLA4 inhibits the expression of the human peripheral blood T cell surface molecule CD25

After the human peripheral blood T cells are stimulated and activated by PHA, the molecular expression on the surface is changed. To exclude interference of CD25 expressed on the surface of NK cells in peripheral blood cells due to low purity of isolated normal human peripheral blood cells, we labeled mature T cells in peripheral blood cells with CD3 antigen by two-color staining method and then observed the expression of CD25 on the surface of mature T cells. The results, expressed as the percentage of CD25 expression positive cells to CD3 positive cells, found that (Anti-CD40mAb) -CTLA4 and CTLA4-Ig significantly inhibited CD25 expression, whereas Anti-CD40mAb did not, as shown in fig. 8.

Example 9: (Anti-CD40mAb) -CTLA4 inhibits human peripheral blood B cell activation

Human Peripheral Blood Mononuclear Cells (PBMC) were used to characterize (Anti-CD40mAb) -CTLA4 ability to affect B cell activation. CD20 expression is selected as an index of B cells, and the expression level of CD80 molecules on the surfaces of the B cells is increased after the B cells are activated. In the experiment, PBMCs from humans were cultured in the presence of 154+ Jurkat Dl.1 cells (immortalized T lymphocyte cell line). B cell activation was determined by measuring the expression of the marker CD80 in CD20+ cells present in PBMCs. Culturing PBMCs in the presence of Jurkat cells resulted in increased expression of all CD80, indicating that B cells are activated by CD154+ Jurkat cells. To test the ability of the antibodies to block B cell activation, PBMCs and Jurkat cells were co-cultured in four groups of treatments (BSA, CTLA4-Ig, Anti-CD40mAb or (Anti-CD40mAb) -CTLA 4). The results show that (Anti-CD40mAb) -CTLA4 significantly reduced CD80 expression with Anti-CD40mAb, blocking B cell activation, whereas CTLA4-Ig failed to block B cell activation as shown in fig. 9.

Example 10: (Anti-CD40mAb) -CTLA4 significantly blocked T cell-dependent Anti-KLH antibody responses

Macaques were immunized once on day 0 with keyhole limpet M hemocyanin (KLH, 10mg IM) antigen (Biosearch Technologies, Novato, Calif.) conjugated to 4-hydroxy-3-nitrophenylacetyl. Prior to vaccination and at 1 week, the experimental macaques received intravenous doses (50mg/kg) of BSA, CTLA4-Ig, Anti-CD40mAb or (Anti-CD40mAb) -CTLA4, respectively. All animals were observed for 70 days and flow cytometry was performed weekly. T cell dependent antibody responses to KLH-NP were tested by ELISA. Plates were coated with KLH (0.01mg/ml, Sigma, St. Louis, Mo.) and blocked with Super Block (Thermoscientific, Woodstock, GA). Plasma samples before and after treatment were serially diluted, plated for 1 hour, and washed with phosphate buffered saline/0.05% Tween. anti-KLH antibodies were detected by incubation with a monoclonal antibody called monkey IgG-horseradish peroxidase (clone 1B3, NHP regentresource, Boston, MA) for 1 hour. The plates were then incubated with peroxidase substrate solution (KPL). Stop solution (KPL) was then added and the optical density was read at 450nm on an ELISA reader. A sample is considered positive at a given dilution if the optical density reading of the plasma after treatment exceeds the optical density of the plasma at the same dilution before treatment by a factor of 2. After KLH immunization, control animals produced high titers of KLH-specific IgG (fig. 10). Animals receiving (Anti-CD40mAb) -CTLA4 also developed Anti-KLH responses, but titers were 20-fold lower than either the CTLA4-Ig group or the Anti-CD40mAb group.

Example 11: significantly prolonged islet allograft survival in a cynomolgus monkey model of xenogenic islet transplantation (Anti-CD40mAb) -CTLA4

We further tested the effect of (Anti-CD40mAb) -CTLA4 on the inhibition of immune rejection in a non-human primate xenograft model. GTKO Wuzhishan minipigs weighing 40kg were subjected to donor pancreatectomy and one day later transplanted via midline laparotomy. Pancreas was isolated after bleeding at the end of the animal and placed on ice. Membrane island separation was performed using collagenase/neutral protease (950 Wunsch units and 63 units, respectively; Serva, Heidelberg, Germany). The digested membrane glands were purified on a four-layer discontinuous euroficol gradient (Mediatech, Manassas, VA) and Cobe 2991 blood cell processor (CaridianBCT, Lakewood, CO). Samples of the final islet preparation were counted and expressed as Islet Equivalents (IEQ). Isolated islets were cultured overnight, counted, and suspended in transplant medium (Mediatech).

5 macaques weighing 3.5kg were made diabetic using streptozotocin (1250mg/m2 IV; Zanosar, Teva Parameral mediines, Irvine, Calif.) and transplanted four weeks later. Diabetes was verified by intravenous glucose tolerance test (IVGTT) with an intravenous bolus of 500mg/kg glucose and measurement of primate C-peptide. Glucose levels were monitored at baseline and 10, 30, 60 and 90 post-glucose injection and C-peptide was measured. Diabetes is confirmed by measuring an increase in blood glucose levels in the absence of detectable serum C-peptide. Diabetic recipients undergo MHC mismatched islet xenotransplantation. Mean 15.745 (soil 4.063) was infused via a small midline laparotomy and mesenteric venous cannula. Blood glucose levels were measured twice daily by ear stick (earstick); NPH (Novolin; novoNordisk, Princeton, N.J.) and Megalotin (Lantus; Sanof1-Aventis, Bridgewater, N.J.) were administered in order to maintain Fasting Blood Glucose (FBG) below 300mg/dL before transplantation and after graft rejection. IVGTT is performed periodically after transplantation to monitor graft function. Transplant recipients were analyzed weekly by flow cytometry to monitor the T cell (CD 3V 450, CD4 PerCP-Cy5.5, CD8 PerCp; BD Bioscience) and B cell (CD20PE, BDbioscience) populations. After islet transplantation, rejection was defined as FBG greater than 130mg/dL on two consecutive days. The primary endpoint was rejection-free membrane island graft survival.

Transplant recipients received 5 groups of CTLA4-Ig, Anti-CD40mAb, (Anti-CD40mAb) -CTLA4, basiliximab (Simulect, Novartis, Basel, Switzerland), and sirolimus (sirolimus). Antibodies (50mg/kg) were administered intravenously on days 0 and 7 (POD) post-surgery. Basiliximab (0.3mg/kg) was administered intravenously at POD 0 and 3. Sirolimus was administered intramuscularly daily to achieve trough levels to POD 1205-15 ng/ml. All three animals receiving basiliximab and sirolimus alone were controls.

Compared to the control, (Anti-CD40mAb) -CTLA4 treatment resulted in significantly prolonged islet graft survival with a median time to graft survival without rejection of 215 days, compared to the CTLA4-Ig treatment of 115 days and the Anti-CD40mAb group of 92 days, as shown in table one.

TABLE-islet survival time (unit: day) in a macaque model of xenogenic islet transplantation

The foregoing is a more detailed description of the present invention that is presented in conjunction with specific embodiments, and the practice of the invention is not to be considered limited to those descriptions. For those skilled in the art to which the invention pertains, several simple deductions or substitutions can be made without departing from the spirit of the invention, and all shall be considered as belonging to the protection scope of the invention.

SEQ ID NO：1

(Anti-CD40mAb) -CTLA4 heavy chain sequence

Anti-CD40 mAb -Linker-CTLA4

MYRMQLLSCIALSLALVTQVQLLQSGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKVIRGSSGWSDAFDIWGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSAMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSD

SEQ ID NO：2

(Anti-CD40mAb) -CTLA4 light chain sequence

MYRMQLLSCIALSLALVTQAVLTQPSSASGTPGQRVTISCSGSSSNIGSHTVSWYQQLPGTAPKLLIYSTDQRPSGVPDRLSGSKSGTSASLTISGLQSEDEAHYYCAAWDDSQNKSLVFGGGTQLTVLGGLGTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO：3

Heavy chain CDR1

SYGMH

SEQ ID NO：4

Heavy chain CDR2

VISYDGSNKYYADSVKGRFT

SEQ ID NO：5

Heavy chain CDR3

VIRGSSGWSDAFDI

SEQ ID NO：6

Light chain CDR1

SGSSSNIGSHTVS

SEQ ID NO：7

Light chain CDR2

STDQRPS

SEQ ID NO：8

Light chain CDR3

AAWDDSQNKSLV

Claims

1. An anti-immune rejection bifunctional molecule, which is expressed by fusing an anti-human CD40 antibody with CTLA4 protein, can simultaneously block CD40-CD154 and CD28-B7 pathways, and has the functions of simultaneously blocking T, B cell activation and inducing immune tolerance;

the CDR regions of the heavy chain of the CD40 antibody consist of VHCDR1, VHCDR2 and VHCDR3, the sequence of the VHCDR1 is SEQ ID NO: 3, the sequence of VHCDR2 is SEQ ID NO: 4, the sequence of VHCDR3 is SEQ ID NO: 5; the CDR regions of the CD40 antibody light chain consist of VLCDR1, VLCDR2 and VLCDR3, and the sequence of the VLCDR1 is SEQ ID NO: 6, the sequence of the VLCDR2 is SEQ ID NO: 7, the sequence of the VLCDR3 is SEQ ID NO: 8;

the CTLA4 protein is directly linked or mediated by a linking sequence to the C-terminus of the heavy chain of an anti-human CD40 antibody.

2. The molecule of claim 1, wherein the anti-human CD40 antibody is of the IgG4 subtype.

3. The molecule of claim 1, wherein said antibody is a monoclonal antibody directed against human CD40 protein and the amino acid sequence formed by the association of CTLA4 with the heavy chain of said antibody is SEQ ID NO: 1, the amino acid sequence of the light chain of said antibody is SEQ ID NO: 2.

4. use of a molecule according to any one of claims 1 to 3 in the manufacture of an antibody medicament for the treatment of transplant rejection or graft-versus-host disease, as well as autoimmune disease.