CN116997570A - anti-CD 38 antibodies for the treatment of antibody-mediated graft rejection - Google Patents

Abstract

The present invention relates to the use of the anti-CD 38 antibody phenanthretomumab in the prevention and/or treatment of graft antibody mediated rejection (ABMR). According to the present invention, the fezetuzumab is effective in treating antibody-mediated renal allograft rejection.

Description

anti-CD 38 antibodies for the treatment of antibody-mediated graft rejection

Technical Field

The present disclosure relates to the field of organ transplantation (e.g., kidney transplantation). In particular, the disclosure relates to anti-CD 38 antibodies for use in treating patients suffering from antibody-mediated transplant rejection (ABMR). The present disclosure provides methods of using anti-CD 38 antibodies to reduce antibody secreting cells and to reduce the level of antibodies specific for one or more antigens present on a transplanted organ. According to the present invention, anti-CD 38 antibodies, alone or in combination with one or more immunosuppressive drugs, may be effective in treating and/or preventing ABMR. The anti-CD 38 antibodies used according to the present invention include phenanthrenetuzumab (MOR 202).

Background

Organ transplantation is a medical procedure in which an organ is removed from the body of a subject (donor) and placed in the body of a recipient (host) to replace the damaged or missing organ. Transplantation is a treatment option for patients with end-stage organ failure. Transplantation (so-called allografts) is performed primarily between two subjects of the same species to reduce organ rejection by the host immune system. However, the host immune system still recognizes well-matched grafts, which may eventually be destroyed.

Previously, alloreactive T cells were considered to be responsible only for graft damage caused by T Cell Mediated Rejection (TCMR). Meanwhile, anti-donor alloantibodies have been identified as another important obstacle to long-term graft survival. This so-called antibody-mediated rejection (ABMR) often results in graft loss (graft loss) after organ transplantation. anti-Donor Specific Antibodies (DSA) such as anti-Human Leukocyte Antigen (HLA) antibodies are major triggers of chronic graft injury, which may be combined with antibody-mediated activation of cellular mechanisms (e.g., activation of natural killer cells). ABMR is one of the major causes of allograft dysfunction and chronic allograft injury in kidney transplantation. Rejection of transplanted kidneys, usually initiated by anti-HLADSA, is associated with progressive decline in Glomerular Filtration Rate (GFR), increased proteinuria, and renal failure.

There are many studies in the prior art to evaluate different therapeutic strategies for ABMR. Known strategies include, for example,

immunosuppression with tacrolimus (tacrolimus), mycophenolate mofetil (mycophenolate mofetil) and belatacept (CTLA-4 Fc-fusion) (Theruvath, TP et al 2001, transformation, 72:77-83;Schwarz,C et al.2015,Transplant International,28:820-827),

Immunomodulation measures, including high doses of intravenous immunoglobulin with or without anti-CD 20 rituximab (rituximab) (Fehr, T et al 2009, translation, 87:1837-1841),

proteasome inhibitor bortezomib (bortezomib) (Walsh, RC et al 2012, kidney Int, 81:1067-1074), or

Complement inhibitors (Eskandary, F et al 2017, am J Transplay, 18:916-926).

However, these strategies do not fully achieve significant improvements over the long term. Thus, there remains a need for improved treatment regimens for long term survival of grafts.

One promising target may be CD38, which is expressed primarily on immune and hematopoietic cells, with particularly high levels of expression on antibody-producing plasma cells. Given the critical role of alloantibody-producing plasma cells in ABMR (when DSA is the cause of injury), efficient plasma cell depletion via CD38 can be used in transplantation medicine to achieve sustained DSA reduction.

The concept of counteracting ABMR with anti-CD 38 antibodies has been presented in the prior art by daratumumab (daratumumab). In the kidney transplant rhesus model, darimumab reduced donor-specific antibodies and caused prolonged survival of the kidney allograft (Kwun, J et al 2019, journal of the American Society of Nephrology, 30:1206-1219). WO2020185672 (celars-Sinai) exemplifies that two patients with anti-HLA antibodies and standard of care tolerance ABMR received darimumab treatment, resulting in a preliminary decrease in anti-HLA antibody levels.

However, a disadvantage of this treatment is the increase in CD4 and CD 8T cells and the elimination of regulatory B cells (B-regs) following treatment with darimumab. This is probably due to the secondary role of darifenacin in regulatory T and B cell depletion. Thus, targeting CD38 with darimumab not only results in a reduction of plasma cell populations, but also depletes beneficial regulatory cell populations. The presence of regulatory T cells (Tregs) in the peripheral circulation and in the graft microenvironment may be important for inducing and maintaining long-term graft tolerance.

Furthermore, there was no meaningful effect on the levels of anti-HLA class II antibodies including DSA DQ5, rebound occurred in several class II antibodies, and HLA class II antibodies reappeared (de novo). This is probably because darimumab is able to deplete cd38+ Natural Killer (NK) cells, thereby limiting ADCC. Figure 1 shows the effect of darifenacin on depletion of NK cells in vitro compared to MOR202 and Ai Satuo ximab.

Overall, these studies effectively control early ABMR attacks, but they demonstrate that the treatment regimens applied in early ABMR have limited effect on late/chronic attacks that remain the primary cause of late graft loss.

Thus, there is a need for new strategies for targeting alloantibody reactivity to treat ABMR and for prolonging long-term graft survival.

The primary modes of action of MOR 202-induced plasma cell lysis are ADCC and ADCP, but not CDC. CDC is believed to be the primary cause of infusion-related reactions. Thus, the main advantage is the lower risk of infusion-related reactions compared to other CD38 antibodies. In addition, MOR202 is depleted primarily of high CD38 cells, thereby retaining specific cell populations with low CD38 levels in vitro. After treatment with MOR202, certain regulatory cell subsets were retained, resulting in improved graft survival.

The present disclosure provides an effective, safe, sustainable and well-tolerated strategy of the anti-CD 38 antibody felzartamab (felzartaab) for managing ABMR, particularly advanced and/or chronic ABMR. Repeated administration of phestuzumab is capable of counteracting tissue inflammation (i.e., an increase in the numbers of cd4+ and cd8+ T cells) and graft damage, particularly inflammation in the microcirculation, B cell responses to HLA antigens, and resultant alloantibody/NK cell-induced chronic graft damage in sustained ABMR.

Disclosure of Invention

The present invention provides anti-CD 38 antibody phenanthretomumab for use in the treatment and/or prevention of organ transplant antibody mediated rejection. Furthermore, the invention provides methods of reducing or eliminating donor-specific antibodies (e.g., anti-HLA) and/or treating or lessening the severity of ABMR in a subject receiving a kidney transplant. The method comprises administering to the patient an effective amount of the anti-CD 38 antibody phenanthrtuzumab. In some aspects, the method further comprises selecting a patient undergoing or having undergone ABMR of organ transplantation. In other aspects, the method further comprises selecting a patient in serum having anti-HLA antibodies specific for the donor HLA.

Drawings

Fig. 1: MOR202 specifically kills MM plasma cell lines that highly express CD38 in vitro, while retaining NK cells that low expression CD38, compared to darimumab (Dara) and Ai Satuo ximab.

Fig. 2: fevertuzumab in advanced ABMR second stage pilot regimen.

Detailed Description

Definition of the definition

The term "CD38" refers to a protein known as CD38, with the following synonyms: ADP-ribose cyclase 1, cADPr hydrolase 1, cycloADP-ribose hydrolase 1, T10.

Human CD38 (UniProt P28907) has the following amino acid sequence:

CD38 is a type II transmembrane glycoprotein, an example of an antigen that is highly expressed on antibody-secreting cells (e.g., plasmablasts and plasma cells). Functions attributed to CD38 include receptor-mediated adhesion and signaling events as well as (extracellular) enzymatic activity. As extracellular enzymes, CD38 uses nad+ as a substrate to form cyclic ADP-ribose (cADPR) and ADPR, as well as nicotinamide and nicotinic acid-adenine dinucleotide phosphate (NAADP). cADPR and NAADP proved to be second messengers of ca2+ movement. By converting nad+ to cADPR, CD38 regulates extracellular nad+ concentration by regulating NAD-induced cell death (NCID), thereby regulating cell survival. In addition to signal transduction by ca2+, CD38 signal transduction also occurs by cross-communication (cross-talk) with antigen-receptor complexes or other types of receptor complexes (e.g. MHC molecules) on T cells and B cells and in this way is involved in several cellular reactions, but also in the conversion and secretion of IgG antibodies.

As used herein, the term "anti-CD 38 antibody" includes the broadest sense of anti-CD 38 binding molecules; including any molecule that specifically binds CD38 or inhibits CD38 activity or function, or that exerts a therapeutic effect on CD38 by any other means. Any molecule that interferes with or inhibits CD38 function is included. The term "anti-CD 38 antibody" includes, but is not limited to, antibodies that specifically bind to CD38, alternative protein scaffolds that bind to CD38, nucleic acids (including aptamers) that are specific for CD38, or small organic molecules that are specific for CD 38.

Antibodies specific for CD38 are described, for example, in WO199962526 (Mayo Foundation), WO200206347 (Crucell Holland), US2002164788 (Jonathan Ellis), WO2005103083, WO2006125640, WO2007042309 (MorphoSys), WO2006099875 (Genmab) and WO2008047242 (Sanofi-Aventis). Combinations of antibodies specific for CD38 and other agents are described, for example, in WO200040265 (Research Development Foundation), WO2006099875 and WO2008037257 (Genmab) and WO2010061360, WO2010061359, WO2010061358 and WO2010061357 (Sanofi Aventis). Antibodies targeting CD38 are widely used in multiple myeloma (reviewed by Frerich KA et al 2018, expert Rev Clin Immunol;14 (3): 197-206). Other uses of anti-CD 38 antibodies are described, for example, in WO2015130732, WO2016089960, WO2016210223 (Janssen), WO2018002181 (UMC Utrecht), WO2019020643 (ENCEFA), WO2020185672 (Cedars-Sinai) and WO2020187718 (MorphoSys). The contents of these documents are incorporated herein by reference in their entirety.

Preferably, the anti-CD 38 antibody for use described herein is an antibody specific for CD 38. More preferably, the anti-CD 38 antibody is an antibody or antibody fragment, e.g., a monoclonal antibody, that specifically binds CD38 and depletes specific CD 38-positive B cells, plasma cells, plasmablasts, and any other CD 38-positive antibody secreting cells. Such antibodies may be of any type, for example, mouse, rat, chimeric, humanized or human.

As used herein, a "human antibody" or "human antibody fragment" is an antibody or antibody fragment having variable regions in which framework and CDR regions are derived from human-derived sequences. If the antibody comprises a constant region, the constant region is also derived from such sequences. Human sources include, but are not limited to, human germline sequences, or mutant forms of human germline sequences, or antibodies comprising consensus framework sequences derived from human framework sequence analysis, e.g., as described by Knappik et al, (2000) J Mol Biol 296:57-86. For example, human antibodies can be isolated from synthetic libraries or transgenic mice (e.g., xenomous). If the sequence of an antibody or antibody fragment is human, the antibody or antibody fragment is human, regardless of the species from which the antibody is physically derived, isolated or prepared.

The structure and position of immunoglobulin variable domains (e.g., CDRs) can be defined using well-known numbering schemes, such as the Kabat numbering scheme, chothia numbering scheme, or a combination of Kabat and Chothia (see, e.g., sequences of Proteins of Immunological Interest, U.S. device of Health and Human Services (1991), eds. Kabatetal.; lazikani et al, (1997) j. Mol. Bio.273:927-948); kabat et al, (1991) Sequences of Proteins of Immunological Interest,5th edition, NIH Publication No.91-3242 U.S.Department of Health and Human Services; chothia et al, (1987) J.mol.biol.196:901-917; chothia et al, (1989) Nature 342:877-883; and Al-Lazikani et Al, (1997) J.mol.biol.273:927-948.

"humanized antibody" or "humanized antibody fragment" is defined herein as an antibody molecule that: it has an antibody constant region and an antibody variable region or portions thereof derived from a human derived sequence, or only CDRs derived from another species. For example, a humanized antibody may be CDR-grafted, in which the CDRs of the variable domain are from a non-human source, while one or more of the frameworks of the variable domain are of human origin and the constant domain (if any) is of human origin.

The term "chimeric antibody" or "chimeric antibody fragment" is defined herein as an antibody molecule that: having antibody constant regions derived from or corresponding to sequences found in one species and antibody variable regions derived from another species. Preferably, the antibody constant region is derived from or corresponds to sequences found in humans, while the antibody variable region (e.g., VH, VL, CDR or FR region) is derived from sequences found in non-human animals such as mice, rats, rabbits, or hamsters.

The term "isolated antibody" refers to an antibody or antibody fragment that is substantially free of other antibodies or antibody fragments having different antigen specificities. Furthermore, the isolated antibody or antibody fragment may be substantially free of other cellular material and/or chemicals. Thus, in certain aspects, antibodies are provided that are isolated antibodies that have been separated from antibodies having different specificities. The isolated antibody may be a monoclonal antibody. The isolated antibody may be a recombinant monoclonal antibody. However, an isolated antibody that specifically binds an epitope, isoform or variant of a target may be cross-reactive with other related antigens (e.g., from other species (e.g., species homologs)).

As used herein, the term "monoclonal antibody" refers to a preparation of antibody molecules having a single molecular composition. Monoclonal antibody compositions exhibit unique binding sites with unique binding specificities and affinities for specific epitopes.

Furthermore, as used herein, "immunoglobulin (Ig)" is defined herein as a protein belonging to the IgG, igM, igE, igA or IgD class (or any subclass thereof), and includes all conventionally known antibodies and functional fragments thereof.

As used herein, the term "antibody fragment" refers to one or more portions of an antibody that retains the ability to specifically interact with an antigen (e.g., by binding, steric hindrance, stable spatial distribution). Examples of binding fragments include, but are not limited to, fab fragments, monovalent fragments consisting of VL, VH, CL and CH1 domains; f (ab) 2 fragments, including two Fab fragments linked at the hinge region by a disulfide bond; fd fragment consisting of VH and CH1 domains; fv fragments consisting of the VL and VH domains of the antibody single arm; a dAb fragment consisting of a VH domain; and an isolated Complementarity Determining Region (CDR). Furthermore, although the two domains of Fv fragments, VL and VH, are encoded by separate genes, they can be joined, using recombinant methods, by a synthetic linker that makes them a single protein chain, wherein the VL and VH regions pair to form a monovalent molecule (referred to as a "single chain fragment" (scFv) such single chain antibodies should be included in the term "antibody fragment"), antibody fragments can also incorporate single-domain antibodies, large antibodies (maxibody), small antibodies (minibody), intracellular antibodies, diabodies (diabody), triabodies (triabody), tetrabodies (tetrabody), v-NAR, and bis-scFv antibody fragments can be grafted onto a polypeptide-based scaffold, such as fibronectin type III (Fn 3).

The present disclosure provides methods of treatment comprising administering to a subject in need of such treatment a therapeutically effective amount of the disclosed anti-CD 38 antibodies. As used herein, "therapeutically effective amount" or "effective amount" refers to the amount of CD 38-specific antibody required to elicit the desired biological response. In accordance with the present disclosure, a therapeutically effective amount is an amount of a CD38 specific antibody required to treat and/or prevent immune complex mediated diseases and symptoms associated with the diseases. The effective amount of a particular individual may vary depending on the condition being treated, the general health of the patient, the route and dosage of the administration, and the severity of the side effects, among other factors (Maynard, et al (1996) A Handbook of SOPs for Good Clinical Practice, intersarm Press, boca Raton, fla.; dent (2001) Good Laboratory and Good Clinical Practice, london, UK).

As used herein, the term "treating" or the like means alleviating symptoms, temporarily or permanently eliminating the cause of symptoms, or preventing or slowing the appearance of symptoms of the disorder or condition. They refer to both therapeutic treatment and prophylactic or preventative measures. The goal of treatment is to prevent or slow (reduce) undesired physiological changes or diseases, or cure the disease to be treated. Beneficial or desired clinical results include alleviation of symptoms, diminishment of extent of disease, stabilization of (i.e., not worsening) the disease state, delay or slowing of disease progression, amelioration or palliation of the disease state, and regression (whether partial or total), whether detectable or undetectable. "treatment" may also mean that the lifetime is prolonged compared to the lifetime expected if the subject is not receiving treatment. Subjects in need of treatment include subjects already with the condition or disorder, subjects prone to have the condition or disorder, or subjects to be prevented from the condition or disorder.

"preventing" refers to reducing the risk of acquiring or developing a disease or disorder (i.e., leaving at least one clinical symptom of the disease non-developing in a subject that may be exposed to a pathogenic agent or susceptible to the disease prior to onset of the disease). "preventing" refers to a method intended to prevent or delay the onset of a disease or symptom thereof.

"administration" includes, but is not limited to, delivery of the drug by injectable forms, e.g., intravenous, intramuscular, intradermal, or subcutaneous routes, or mucosal routes, e.g., as nasal sprays or aerosols for inhalation, or as ingestible solutions, capsules, or tablets. Preferably, the administration is in injectable form.

Any means of delivering two or more therapeutic agents to a patient by co-administration, including as part of the same therapeutic regimen, will be apparent to the skilled artisan. Although two or more agents may be administered simultaneously in a single formulation, i.e., as a single pharmaceutical composition, this is not required. The agents may be administered at different times in different formulations. Therapies (e.g., prophylactic or therapeutic agents) of the combination therapy can be administered to a subject concomitantly (simultaneously) or sequentially. Therapies (e.g., prophylactic or therapeutic agents) of the combination therapy may also be administered periodically. Cycling therapy includes administering a first therapy (e.g., a first prophylactic or therapeutic agent) for a period of time, then administering a second (e.g., a second prophylactic or therapeutic agent) therapy for a period of time, and repeating such sequential administration, i.e., cycling. This is to reduce tolerance to one of the therapies, to avoid or reduce side effects of one of the therapies, and/or to increase the efficacy of these therapies. The term "concomitant" or "simultaneous" is not limited to administration of therapies at exactly the same time, but rather means that a pharmaceutical composition comprising an antibody or antibody fragment of the disclosure is administered to a subject sequentially and over a time interval such that the antibodies of the disclosure can act with other therapies to provide greater benefit than administering them in other ways.

As used herein, "subject" or "species" refers to any mammal, including rodents (e.g., mice or rats), as well as primates, such as cynomolgus monkey (Macaca fascicularis), rhesus monkey (Macaca mulatta), or human (Homo sapiens). Preferably, the subject is a primate, most preferably a human.

As used herein, the term "subject in need thereof" and the like refers to a human or non-human animal patient that exhibits one or more symptoms or signs of organ graft antibody-mediated rejection. Preferably, the subject is a primate, most preferably a human patient who has been diagnosed with antibody-mediated rejection following a kidney transplant.

The term "antibody-mediated rejection" ("ABMR") refers to the resulting defined entity, which often occurs after organ transplantation (Tx), includes diagnostic criteria defined according to the Banff classification, e.g. inflammation and morphological damage in the microcirculation, deposition of complement cleavage product C4d along the transplanted endothelium (optional), and detection of antibodies against donor antigens ("donor-specific antibodies", DSA). DSAs may be (i) antibodies against donor HLA and/or (ii) non-HLA antibodies, which may be classified into at least two main classes: an alloantibody against a polymorphic antigen that differs between a recipient and a donor, and an antibody or autoantibody that recognizes an autoantigen.

As used herein, the term "about" when used with respect to a particular recited value means that the value may differ from the recited value by no more than 1%. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

By "pharmaceutically acceptable" is meant approved or approvable by a regulatory agency of the federal or a state government or a corresponding agency in a country other than the united states, or listed in the united states pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

"pharmaceutically acceptable vehicle" refers to a diluent, adjuvant, excipient, or carrier with which an antibody or antibody fragment is administered.

Throughout this specification, unless the context requires otherwise, the words "comprise," "have" and "comprise" and their respective variations such as "comprises," "comprising," "having," "including," "containing" and "comprising" will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

"Pheretima" is an anti-CD 38 antibody, also known as "MOR202", "MOR03087", or "MOR3087". These terms may be used interchangeably throughout this disclosure. MOR202 has an IgG1Fc region.

According to Kabat, the amino acid sequence of MOR202HCDR1 is:

SYYMN(SEQ ID NO:1)

according to Kabat, the amino acid sequence of MOR202HCDR2 is:

GISGDPSNTYYADSVKG(SEQ ID NO:2)

according to Kabat, the amino acid sequence of MOR202HCDR3 is:

DLPLVYTGFAY(SEQ ID NO:3)

according to Kabat, the amino acid sequence of MOR202LCDR 1 is:

SGDNLRHYYVY(SEQ ID NO:4)

according to Kabat, the amino acid sequence of MOR202LCDR2 is:

GDSKRPS(SEQ ID NO:5)

according to Kabat, the amino acid sequence of MOR202LCDR 3 is:

QTYTGGASL(SEQ ID NO:6)

the amino acid sequence of the MOR202 variable heavy domain is:

the amino acid sequence of the MOR202 variable light chain domain is:

the DNA sequence encoding the MOR202 variable heavy domain is:

the DNA sequence encoding the MOR202 variable light chain domain is:

description of the embodiments

Antibodies to

In certain embodiments of the present disclosure, antibodies or antibody fragments specific for CD38 used in accordance with the present disclosure comprise a variable heavy chain variable region, a variable light chain region, a heavy chain, a light chain, and/or CDRs comprising any of the amino acid sequences of the CD38 specific antibodies described in WO 2007042309.

In one embodiment, an antibody or antibody fragment specific for CD38 for use in accordance with the present disclosure comprises: an HCDR1 region comprising the amino acid sequence of SEQ ID NO. 1, an HCDR2 region comprising the amino acid sequence of SEQ ID NO. 2, an HCDR3 region comprising the amino acid sequence of SEQ ID NO. 3, an LCDR1 region comprising the amino acid sequence of SEQ ID NO. 4, an LCDR2 region comprising the amino acid sequence of SEQ ID NO. 5, and an LCDR3 region comprising the amino acid sequence of SEQ ID NO. 6.

In one embodiment, an antibody or antibody fragment specific for CD38 for use in accordance with the present disclosure comprises: the HCDR1 region of SEQ ID NO. 1, the HCDR2 region of SEQ ID NO. 2, the HCDR3 region of SEQ ID NO. 3, the LCDR1 region of SEQ ID NO. 4, the LCDR2 region of SEQ ID NO. 5, and the LCDR3 region of SEQ ID NO. 6.

In one embodiment, an antibody or antibody fragment specific for CD38 for use in accordance with the present disclosure comprises: the variable heavy chain region of SEQ ID NO. 7 and the variable light chain region of SEQ ID NO. 8.

In another embodiment, an anti-CD 38 antibody or antibody fragment for use according to the present disclosure comprises: the variable heavy chain region of SEQ ID NO. 7 and the variable light chain region of SEQ ID NO. 8, or the variable heavy chain region and the variable light chain region having at least 60%, at least 70%, at least 80%, at least 90% or at least 95% identity to the variable heavy chain region of SEQ ID NO. 7 and the variable light chain region of SEQ ID NO. 8.

Exemplary antibodies or antibody fragments for use according to the present disclosure comprise: the variable heavy chain region comprising the amino acid sequence of SEQ ID NO. 7 and the variable light chain region comprising the amino acid sequence of SEQ ID NO. 8 are human anti-CD 38 antibodies known as MOR202 (Pheretima mab).

In one embodiment, the present disclosure relates to a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an antibody or antibody fragment specific for CD38 for use according to the present disclosure, wherein the antibody or antibody fragment comprises the HCDR1 region of SEQ ID No. 1, the HCDR2 region of SEQ ID No. 2, the HCDR3 region of SEQ ID No. 3, the LCDR1 region of SEQ ID No. 4, the LCDR2 region of SEQ ID No. 5, and the LCDR3 region of SEQ ID No. 6.

In another embodiment, the disclosure relates to a nucleic acid encoding an isolated monoclonal antibody or fragment thereof for use according to the disclosure, wherein the nucleic acid comprises the VH of SEQ ID No. 10 and the VL of SEQ ID No. 11.

In one embodiment, the antibodies or antibody fragments disclosed for use in accordance with the present disclosure that are specific for CD38 are monoclonal antibodies or antibody fragments.

In one embodiment, the disclosed antibodies or antibody fragments specific for CD38 used in accordance with the present disclosure are human, humanized or chimeric antibodies.

In certain embodiments, the antibody or antibody fragment specific for CD38 used in accordance with the present disclosure is an isolated antibody or antibody fragment.

In another embodiment, the antibody or antibody fragment used according to the present disclosure is a recombinant antibody or antibody fragment.

In other embodiments, the antibodies or antibody fragments used according to the present disclosure are recombinant human antibodies or antibody fragments.

In other embodiments, the recombinant human antibody or antibody fragment used according to the present disclosure is an isolated recombinant human antibody or antibody fragment.

In other embodiments, the recombinant human antibody or antibody fragment or isolated recombinant human antibody or antibody fragment used according to the present disclosure is monoclonal.

In one embodiment, the disclosed antibodies or antibody fragments for use according to the present disclosure are of the IgG isotype. In a particular embodiment, the antibody is IgG1.

In a particular aspect of the invention, the anti-CD 38 antibody used according to the present disclosure is MOR202 (fezetuzumab).

In one embodiment, the present disclosure relates to a pharmaceutical composition for use according to the present disclosure comprising a phenanthrenetuzumab (MOR 202) or fragment thereof, which is specific for CD38, and a pharmaceutically acceptable carrier or excipient.

In certain embodiments, the antibody or antibody fragment specific for CD38 is an isolated monoclonal antibody or antibody fragment that specifically binds human CD 38.

Pharmaceutical composition

When used as a medicament, the CD38 specific antibody or antibody fragment is typically administered in a pharmaceutical composition. The compositions of the present disclosure are preferably pharmaceutical compositions comprising phenanthretomumab (MOR 202) and a pharmaceutically acceptable carrier, diluent or excipient for use in treating, inhibiting and/or reducing the severity of organ transplant antibody mediated rejection (ABMR) reactions in a subject in need thereof.

The pharmaceutically acceptable carrier should be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion).

The pharmaceutically acceptable carrier enhances or stabilizes the composition, or facilitates the preparation of the composition. Pharmaceutically acceptable carriers include physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. In many cases, it is preferred to include isotonic agents, for example, sugars, polyalcohols (e.g., mannitol or sorbitol) and sodium chloride in the composition.

The pharmaceutical compositions of the present disclosure may be administered by a variety of routes known in the art. The route of administration selected for the antibodies or antibody fragments of the present disclosure includes intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, for example by injection or infusion.

Antibodies or antibody fragments specific for CD38 are preferably formulated as injectable compositions. In a preferred aspect, the anti-CD 38 antibodies of the present disclosure are administered intravenously. In other aspects, the anti-CD 38 antibodies of the disclosure are administered subcutaneously, intra-articular, or intrathecally.

An important aspect of the present disclosure is a pharmaceutical composition capable of mediating killing of antibody-secreting cells (e.g., plasmablasts, plasma cells) expressing CD38 by ADCC and ADCP.

Therapeutic method

In one embodiment, the present disclosure provides an anti-CD 38 antibody or antibody fragment or a pharmaceutical composition comprising an anti-CD 38 antibody or antibody fragment for treating, inhibiting and/or reducing the severity of an organ transplant antibody-mediated rejection (ABMR) response in a subject in need thereof.

In certain embodiments, the organ transplant is one or more of kidney, heart, liver, lung, pancreas, stomach, skin, and intestine.

In one embodiment, the present disclosure provides an anti-CD 38 antibody or antibody fragment or a pharmaceutical composition comprising an anti-CD 38 antibody or antibody fragment for treating, inhibiting and/or reducing the severity of a renal graft antibody-mediated rejection (ABMR) response in a subject in need thereof.

In a particular embodiment, the present disclosure provides an anti-CD 38 antibody or antibody fragment comprising the HCDR1 region of SEQ ID No. 1, the HCDR2 region of SEQ ID No. 2, the HCDR3 region of SEQ ID No. 3, the LCDR1 region of SEQ ID No. 4, the LCDR2 region of SEQ ID No. 5 and the LCDR3 region of SEQ ID No. 6 for use in treating, inhibiting and/or reducing the severity of an organ transplant antibody mediated rejection (ABMR).

In another aspect, the present disclosure provides an anti-CD 38 antibody or antibody fragment comprising the variable heavy chain region of SEQ ID No. 7 and the variable light chain region of SEQ ID No. 8 for use in treating, inhibiting and/or reducing the severity of an organ graft antibody mediated rejection (ABMR) response in a subject in need thereof.

In a particular aspect, the present disclosure provides MOR202 (fezetuzumab) for treating, inhibiting, and/or reducing the severity of an organ graft antibody mediated rejection (ABMR) response in a subject in need thereof.

In one embodiment, the present disclosure provides an anti-CD 38 antibody or antibody fragment for depleting CD38 expressing antibody secreting cells (preferably plasma cells) in a subject having an antibody-mediated rejection (ABMR) response following organ transplantation.

In a preferred embodiment, the present disclosure provides an anti-CD 38 antibody (e.g., MOR 202) for reducing circulating anti-HLA antibodies and/or anti-non-HLA antibodies in a subject having an antibody-mediated rejection (ABMR) response following an organ transplant.

In another embodiment, the present disclosure provides an anti-CD 38 antibody (e.g., MOR 202) for reducing deposited anti-HLA and/or anti-non-HLA antibodies in transplanted organs in a subject having an antibody-mediated rejection (ABMR) response following organ transplantation.

In a further aspect, the present disclosure provides a therapeutic agent comprising an anti-CD 38 antibody (e.g., MOR 202) as an active ingredient for alleviating a symptom of ABMR in a subject who has received a kidney transplant, wherein the symptom is selected from the group consisting of (i) worsening renal function as measured by serum creatinine and estimated glomerular filtration rate (evfr); (ii) the presence of a donor-specific antibody; and/or (iii) capillary inflammation, inflammation and complement (C4 d) deposition in the kidney.

In another aspect, the present disclosure provides a prophylactic and/or therapeutic agent comprising an anti-CD 38 antibody (e.g., MOR 202) for restoring, improving, or normalizing kidney function as indicated by glomerular filtration rate (eGFR) based on the CKD-epi equation in a subject having an antibody-mediated rejection (ABMR) response following a kidney transplant.

In a further aspect, the present disclosure provides an anti-CD 38 antibody (e.g., MOR 202) for use in treating an organ transplant ABMR response in a subject in need thereof, wherein the anti-CD 38 antibody (e.g., MOR 202) is dosed in at least 2, at least 5, at least 7, or at least 9 doses.

In another aspect, the present disclosure provides an anti-CD 38 antibody (e.g., MOR 202) for use in treating an organ transplant ABMR response in a subject in need thereof, wherein the anti-CD 38 antibody (e.g., MOR 202) is dosed at 2, 5, 7, or 9 doses.

In a specific embodiment, the dosage is 8 mg/kg or higher. In a particular embodiment, the dose is 16mg/kg.

In another embodiment, the present disclosure provides an anti-CD 38 antibody for use in treating organ transplant ABMR in a subject in need thereof, wherein the antibody is administered in the following manner: every two weeks in cycle 1 (C1), and every 4 weeks in cycles 2-6 (administration of fevertuzumab/placebo on days 0 and 14 (cycle 1), followed by 4 weeks at intervals of 4 weeks (cycles 2-6) on weeks 4, 8, 12, 16 and 20).

In another embodiment, the present disclosure provides an anti-CD 38 antibody for use in treating ABMR, wherein the anti-CD 38 antibody is administered intravenously.

In another embodiment, the disclosure provides an anti-CD 38 antibody for use in treating ABMR, wherein the antibody is administered intravenously over a period of two hours.

In one embodiment, the anti-CD 38 antibody (e.g., MOR 202) is administered prior to, concurrently with, and/or after organ transplantation.

In another embodiment, the present disclosure provides a method of treating a subject in need of transplantation by administering to the subject an effective amount of fevertuzumab prior to, concurrently with and/or after transplantation.

In another aspect, the present disclosure provides the use of an anti-CD 38 antibody or antibody fragment in the manufacture of a medicament for treating and/or preventing organ transplant antibody-mediated rejection (ABMR) response in a subject in need thereof.

In other aspects, the present disclosure provides the use of an anti-CD 38 antibody or antibody fragment comprising the HCDR1 region of SEQ ID NO:1, the HCDR2 region of SEQ ID NO: 2, the HCDR3 region of SEQ ID NO: 3, the LCDR1 region of SEQ ID NO: 4, the LCDR2 region of SEQ ID NO:5 and the LCDR3 region of SEQ ID NO:6 in the manufacture of a medicament for treating and/or preventing organ graft antibody mediated rejection (ABMR) in a subject in need thereof.

In other aspects, the present disclosure provides the use of an anti-CD 38 antibody or antibody fragment comprising the variable heavy chain region of SEQ ID NO:7 and the variable light chain region of SEQ ID NO:8 in the manufacture of a medicament for treating and/or preventing organ graft antibody mediated rejection (ABMR) reactions in a subject in need thereof.

In a further aspect, the present disclosure provides the use of MOR202 (fezetuzumab) in the manufacture of a medicament for treating and/or preventing a renal graft antibody-mediated rejection (ABMR) response in a subject in need thereof.

In other aspects, the present disclosure provides for the use of MOR202 (fezetuzumab) or a pharmaceutical composition comprising MOR202 (fezetuzumab), in combination with another therapeutic agent, in the manufacture of a medicament for treating and/or preventing an organ graft antibody-mediated rejection (ABMR) response in a subject in need thereof.

In some embodiments, the present disclosure provides the use of MOR202 in combination with a steroid for the treatment and/or prevention of ABMR in a subject in need thereof. In other aspects, MOR202 is administered in combination with a proteasome inhibitor, such as bortezomib (bortezomib) or carfilzomib (carfilzomib), for use in the treatment and/or prevention of ABMR.

In one aspect, the present disclosure provides a method for treating and/or preventing an organ transplant antibody mediated rejection (ABMR) response in a subject in need thereof, comprising administering to the subject an anti-CD 38 antibody. In certain embodiments, the antibody-mediated rejection (ABMR) response is against a kidney graft.

In some embodiments, the disclosure provides methods for preventing and/or treating a subject having an organ transplant antibody mediated rejection (ABMR) response, wherein the subject is resistant to treatment by other immunosuppressive therapies including corticosteroids or calcineurin inhibitors or B cell depletion therapies (e.g., using rituximab or any other anti-CD 20 antibody or anti-BAFF antibody), comprising administering an effective amount of an anti-CD 38 antibody or antibody fragment.

In one aspect, the present disclosure provides methods of using an anti-CD 38 antibody or antibody fragment to achieve a prophylactic or therapeutic benefit in a patient susceptible to or affected by an antibody-mediated rejection (ABMR) response following an organ transplant.

In another aspect, the present disclosure provides a method for reducing the incidence of, ameliorating the effects of, inhibiting the effects of, alleviating the effects of, and/or delaying the onset, progression, or progression of an antibody-mediated rejection (ABMR) response in a subject, the method comprising administering to the subject an effective amount of an anti-CD 38 antibody. In particular, antibody-mediated rejection (ABMR) responses are after kidney transplantation.

In preferred embodiments, the present disclosure provides methods for treating patients with elevated DSA levels associated with antibody-mediated rejection (ABMR) responses.

In other aspects, the disclosure provides methods for treating and/or preventing diseases caused by the presence of donor-specific antibodies. In other aspects, the disclosure provides methods for treating and/or preventing symptoms associated with the presence of anti-donor HLA antibodies. In a further aspect, the present disclosure provides methods for treating and/or preventing symptoms associated with the presence of anti-donor antibodies that are not directed against HLA.

In other embodiments, the disclosure provides methods of reducing inflammation and C4d complement deposition in a subject having antibody-mediated rejection (ABMR), the method comprising administering an effective amount of an anti-CD 38 antibody or antibody fragment described herein or one or more pharmaceutical compositions. For example, the methods provided herein comprise administering an anti-CD 38 antibody to a patient having an elevated level of an anti-HLA antibody. In other aspects, the methods provided herein comprise administering an anti-CD 38 antibody to a patient having an elevated level of C4d complement deposition in a transplanted organ.

In one embodiment, the decrease (change) in anti-HLA levels in serum of a subject having antibody-mediated rejection (ABMR) is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% compared to baseline following administration of an anti-CD 38 antibody or antibody fragment or one or more pharmaceutical compositions described herein.

In another aspect, the present disclosure provides a method for preventing reduced renal function in an individual having antibody-mediated rejection (ABMR), the method comprising administering an effective amount of an anti-CD 38 antibody or antibody fragment described herein or one or more pharmaceutical compositions.

In a further embodiment, the disclosure relates to a method for treating antibody-mediated rejection (ABMR) in a subject, comprising administering to the subject a pharmaceutical composition comprising an anti-CD 38 antibody or antibody fragment that binds CD38 expressing cells and depletes these CD38 expressing cells.

In a preferred embodiment, the present disclosure relates to a method for treating ABMR in a subject comprising administering to the subject a pharmaceutical composition comprising an anti-CD 38 antibody or antibody fragment that binds to and depletes antibody secreting cells expressing CD38 while retaining regulatory T cells and/or B cell populations.

In another embodiment, the present disclosure relates to a method for treating ABMR in a subject comprising administering to the subject a pharmaceutical composition comprising an anti-CD 38 antibody or antibody fragment that binds to an antibody-secreting cell expressing CD38 and depletes the antibody-secreting cell, but does not result in a significant decrease in regulatory T cells.

In a particularly preferred embodiment, the present disclosure relates to a method for treating antibody-mediated rejection (ABMR) in a subject, comprising administering to the subject a pharmaceutical composition comprising an anti-CD 38 antibody or antibody fragment that binds to CD38 expressing antibody-secreting cells and depletes these CD38 expressing antibody-secreting cells, wherein the specific cell killing effect of the antibody on the antibody-secreting cells is significantly higher than the specific cell killing effect on NK cells.

In one embodiment, the disclosure relates to a method for treating antibody-mediated rejection (ABMR) in a subject, comprising administering to the subject a pharmaceutical composition comprising an anti-CD 38 antibody or antibody fragment that binds to CD38 expressing antibody-secreting cells and depletes these CD38 expressing antibody-secreting cells, wherein the specific cell killing of antibody-secreting cells is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, and wherein the specific cell killing of non-antibody secreting NK cells is 30% or less, 25% or less, 20% or less, as determined by a standard ADCC assay.

In one embodiment, the present disclosure relates to a method for treating antibody-mediated rejection (ABMR) in a subject, comprising administering to the subject a pharmaceutical composition comprising an anti-CD 38 antibody or antibody fragment, wherein the subject has undergone standard-of-care treatment comprising one or more of immunoglobulin administration (IVIG), rituximab administration, and Plasmapheresis (PLEX), and the subject's response to the standard-of-care treatment is ineffective.

In another embodiment, the subject to be treated is further resistant or has acquired resistance to immunosuppressive treatment with one or more of eculizumab (ecalizumab), thymalglobulin (thymobulin), bortezomib, carfilzomib, basiliximab (basiliximab), mycophenolate mofetil, tacrolimus, and corticosteroids.

In another embodiment, the disclosure relates to a method for treating antibody-mediated rejection (ABMR) in a subject, comprising administering to the subject a pharmaceutical composition comprising an anti-CD 38 antibody or antibody fragment, wherein the subject has not undergone any prior standard-of-care (standard-of-care) treatment.

In another embodiment, the present disclosure relates to a method for treating ABMR in a subject comprising administering to the subject a pharmaceutical composition comprising an anti-CD 38 antibody or antibody fragment, wherein administration of the anti-CD 38 antibody does not result in a significant change in the absolute number of regulatory cd4+, cd25+, CD127-T cells.

In another embodiment, the disclosure relates to a method for treating ABMR in a subject comprising administering to the subject a pharmaceutical composition comprising an anti-CD 38 antibody or antibody fragment, wherein the cd8+ T cell/Treg ratio does not increase significantly after antibody administration.

In another embodiment, the disclosure relates to a method for treating ABMR in a subject comprising administering to the subject a pharmaceutical composition comprising an anti-CD 38 antibody or antibody fragment, wherein administration of the anti-CD 38 antibody or antibody fragment results in a decrease in class I and/or class II anti-HLA antibody levels. anti-HLA class I antibodies include anti-HLA-A, -B and-C. anti-HLA class II antibodies include anti-HLA-DR, -DQ (e.g., anti-DQ 5), and-DP.

In a preferred embodiment, the method of treating ABMR is performed in a human subject and comprises administering a pharmaceutical composition comprising MOR202 (fezetuzumab), wherein said administration results in a reduction in the level of class II anti-HLA antibodies, preferably anti-DQ 5 antibodies. In a preferred aspect, 9 doses of MOR202 are administered.

Examples

Example 1 Pheretima mab in advanced antibody-mediated renal allograft rejection

1.1 study design

The present study is a pilot trial driven by researchers aimed at assessing safety, tolerability, pharmacokinetics, pharmacodynamics and efficacy of the fully human anti-CD 38 monoclonal antibody fezetuzumab in renal transplant recipients with advanced or chronic active ABMR.

The test was designed as a randomized, control, double-blind phase 2 pilot test. The primary endpoints are safety and tolerability. A simplified flow chart of the test is shown in figure 2.

1.2 study population

About 20 kidney transplant recipients were included who had circulating anti-HLA DSA and were biopsied at indication (index biopsy; slow deterioration and in clinical routine for positive post-transplant DSA outcome and allograft functionAnd/or proteinuria) with advanced (180 days post-transplant) active ABMR biopsy features (according to Banff2019 protocol). Other key inclusion criteria are: functional grafts with age > 18 years, 180 days after implantation, estimated GFR (eGFR according to CKD-EPI formula) > 20ml/min/1.73m ² . The inclusion and exclusion criteria are detailed in table 1.

Table 1: inclusion and exclusion criteria.

1.3 dosing

Subjects were randomized to receive either fezetuzumab (16 mg/kg, i.v. dosing) or placebo at a 1:1 ratio using a network-based randomization platform (www.meduniwien.ac.at/randomizer). From PK modeling results of the ongoing autoimmune disease stage Ib/IIa trial (membranous nephropathy, clinicalTrials.gov, NCT 04145440), patients were dosed with fevertuzumab by intravenous infusion for 6 months. Since (as opposed to membranous kidney disease patients) transplant patients are receiving multi-compound immunosuppressive baseline therapy, and thus the risk of infection increases, it is planned to extend the dosing interval of the first cycle (every 2 weeks instead of every week). 7 doses, but not 9 doses of fezetuzumab, were administered at 16mg/kg i.v. for 6 treatment cycles of 28 days each. Dosing every two weeks in cycle 1 (C1), dosing every 4 weeks in cycles 2-6 (administration of feveruzumab/placebo on day 0 and day 14 (cycle 1), followed by dosing at 4 weeks intervals on weeks 4, 8, 12, 16 and 20 (cycles 2-6).

In a preferred setting, subjects received either selzetuzumab (16 mg/kg, intravenous administration) or placebo (0.9% saline) (1:1 randomization) at random for a period of 6 months (administration of selzetuzumab/placebo on days 0, 7, 14, 21 (cycle 1), followed by administration at 4 weeks (cycle 2-6) at 4, 8, 12, 16 and 20 weeks (cycle 2-6)) and 12 months after 6 months (24 weeks) and after 12 months (52 weeks), the study participants received subsequent allograft biopsies the main objective of the test was to evaluate the safety, pharmacokinetics and pharmacodynamics (peripheral blood PC and NK cell depletion) of the course of 6 months within 12 months.

Thus, 9 doses of either fezetuzumab or placebo are administered as an intravenous infusion, for 6 treatment cycles of 28 days each. In cycle 1, weekly doses are given, and in cycles 2-6, every 4 weeks.

After reconstitution with 4.8mL of water for injection (one vial containing 325mg of MOR 202), the fevertuzumab was supplied at 65mg/mL in 10mM histidine, 260mM sucrose, 0.1% Tween20 (pH 6.0). Pheretima was administered after dilution with 250mL of 0.9% sodium chloride solution (final concentration should be 1-20 mg/mL). Placebo (0.9% sodium chloride) was infused with 250mL of physiological saline. The prepared infusion may be stored at 2 ℃ to 8 ℃ for up to 24 hours and at room temperature, 15 ℃ to 25 ℃ for up to 4 hours for 24 hours. Prior to administration, the fezeitumumab/placebo infusions must be stored for 30-60 minutes to room temperature by non-refrigerated storage prior to use.

The first two infusions of fevertuzumab were slow (about 90 minutes) and if no infusion reaction occurred, the infusion time in subsequent infusions could be shortened to 1 hour or less (a minimum of 30 minutes).

Study participants received subsequent allograft biopsies after 6 months (week 24) and 12 months (week 52). Randomization was based on ABMR categories (active ABMR versus chronic/active ABMR) to ensure balance of patients with both histological types between the two groups. The study was designed as a double blind trial to minimize bias.

Pre-use medicine (prediction)

To prevent infusion-related reactions, patients assigned to the fezetuzumab group received i.v. pre-medication prior to the first two fezetuzumab infusions (day 0 and day 14). The placebo group of patients received placebo (0.9% nacl solution). The pre-drug was administered 30 minutes prior to the infusion of the fezetuzumab, consisting of Diphenhydramine (30 mg), paracetamol (1000 mg) and prednisolone (Prednisolon, 100 mg) respectively (all 100mL volumes). In the placebo group, the patients received 3X 100mL NaCl 0.9%.

The following drugs were disabled during the study:

rituximab, eculizumab, proteasome inhibitors, IVIG, plasmapheresis or immunoadsorption, other research drugs/treatments, including commercially available CD38 or anti-IL-6/sIL-6R monoclonal antibody drugs, e.g., darimumab Or Torulizumab (Tocilizumab)>

The following concomitant medications were allowed during the study:

calcineurin inhibitor (CNI, tacrolimus or cyclosporine a), mammalian target of rapamycin (mTOR) inhibitor (everolimus or rapamycin), mycophenolate Mofetil (MMF)/mycophenolate sodium; chronic treatment with low doses of corticosteroid (prednisolone 5 mg/day).

Baseline immunosuppression: at the time of diagnosis of advanced ABMR, all recipients treated with calcineurin inhibitors [ tacrolimus or cyclosporine a (CyA) ] or mTOR inhibitors (everolimus or rapamycin) received mycophenolate esters (or, alternatively, enteric coated mycophenolic acid (EC-MPA), at an initial dose of 2 x 500mg per day (or 2 x 360mg respectively) without azathioprine (azathioprine) or mycophenolic acid (MPA), if tolerised, stepwise up to 2 x 1000mg per day (or 2 x 720 mg) to avoid immunosuppression. Tacrolimus was adjusted to achieve a target trough (trough) level of 5-10ng/mL and CyA was adjusted to a target trough level of 80-120 ng/mL. Recipients who discontinued steroid receive low doses of prednisolone (5 mg/day).

1.4 evaluation of efficacy

The main objective of the trial was to evaluate the safety, pharmacokinetics and pharmacodynamics (peripheral blood PC and NK cell depletion) of the 6 month course of treatment over 12 months. In addition, data will be provided regarding efficacy (progression/activity of rejection, blood biomarkers) and potential correlation of treatment with parameters reflecting clinical progression of allograft dysfunction (e.g., renal function processes as described in Irish, W et al 2020, transformation, or iBOX score Loupy, A et al, BMJ,366: l4923, 2019).

TABLE 2 study endpoint

Primary endpoints (see table 2) include safety and tolerability, the course of DSA (and total Ig and IgG subclass levels in parallel), peripheral blood count dynamics (assessed by FACS) of PC, NK cells and T and B cell subsets, as well as rejection biomarkers (CXCL 9 and CXCL10 in blood and urine) and overall immunosuppression (fine ring viral load). In addition, morphology (Banff criteria for rejection and chronic injury; immunohistochemistry for detection of complement activation/deposition and characterization of cell infiltrates including NK cells) and Molecular rejection criteria (Molecular ABMR score; molecular use) of kidney allograft biopsies for 6 months and 12 months were evaluatedMicroarray analysis by diagnostic System) including pathogenesis-based transcript (PBT) scores (cytotoxic T cell infiltration, gamma interferon effect, natural killer cell loading, epithelial cell damage) in 6 months and 12 months biopsies. Clinical endpoints are proteinuria as well as the gfr slope and iBox clinical prediction score, both of which are validated surrogate endpoints that accurately predict long term survival of allografts.

Example 2 Experimental methods

2.1HLA antibody detection

To assess HLA antibody levels, serum samples were assessed following published protocols (dober, K et al; J Am Soc Nephrol) after completion of the study. Briefly, antibody detection was performed using a LABscreen single antigen bead assay (OneLambda). Serum samples were incubated with 10mM EDTA to prevent complement interference. Data acquisition was performed by a LABScanTM200 flow analyzer (Luminex Corporation). For longitudinal analysis of DSA/HLA antibody levels, bead assays were performed retrospectively to avoid the effects of daily variation in test results. Donor specificity is defined in terms of serology and/or low or high resolution donor/acceptor HLA typing (HLA-a, -B, -Cw, -DR, -DQ, -DP). The test results are reported as the Mean Fluorescence Intensity (MFI) of immunodominant DSA. MFI threshold > 1,000 was considered positive. The effect of fevertuzumab treatment on DSA levels was estimated by the percent change in MFI. To more accurately quantify DSA level changes, additional dilution experiments were performed as described by dober K et al 2020, transplating. Briefly, a non-linear standard curve based on the original DSAMFI level (immunodominant DSA) was obtained by serial dilution of individual patient serum collected before the start of treatment (all samples incubated with EDTA) and 24 weeks. The fold change in antibody levels was then calculated from the DSAMFI levels detected in the same experiment for undiluted week 12, 24 and 52 samples, according to the calculated standard curve.

2.2 immunoglobulin levels

Applying immunonephelometry (immunonephelometry) to BN ^TM Total IgG, igM and IgG subclasses were assessed in serum on an analyzer (Siemens Healthineers).

2.3 graft biopsies

Subsequent biopsies were taken at week 24 and week 52 (study end visit) after exclusion of coagulation disorders or platelet counts below 80%. Biopsies are performed under local anesthesia (lidocaine) using ultrasound guided percutaneous techniques. Histomorphometric assessment was performed on paraffin-embedded sections using standard methods. The embedded tissue pieces were subjected to serial sections (5-mm thick) and hematoxylin eosin and periodic acid-schiff staining for routine evaluation and rejection fractionation. For immunohistochemical C4D staining, a polyclonal anti-C4D antibody (BI-RC 4D, biomedica) was used and minimal immunohistochemical staining along perivascular capillaries (C4 dBanff score. Gtoreq.1) was considered positive following the Banff protocol (Loupy, aet al 2020, american Journal of Transplantation: ajt.15898). In addition, biopsy evaluation was performed by electron microscopy, and multilayering of capillary basement membrane around the test tube (MLPTC). In addition, molecular using International authenticationDiagnostic System MMDx platform all biopsies were analyzed using a microarray also proposed by the Banff protocol. Generation of thoroughly validated molecular scores using a reference set of 1529 biopsies [ ABMR, T Cell Mediated Rejection (TCMR), total rejection ] ]Inflammation (global interference score) or chronic injury (atrophy/fibrosis score), based on a classifier based on machine learning-derived lesions associated with rejection. For ABMR classification according to the Banff 2019 protocol, all biopsy results were analyzed in the context of molecular results. ABMR is defined in terms of morphology (histomorphology, immunohistochemistry, electron microscopy) and thoroughly validated molecular criteria: (i) Evidence of acute or chronic tissue injury, (ii) evidence of current/recent antibody interactions with vascular endothelium; and (iii) serological evidence of DSA.

2.4 renal function

Evaluation of eGFR was performed using the chronic kidney disease epidemiological collaboration (CKD-EPI) equation (mL/min/1.73 m ² ). Protein excretion was recorded as the protein/creatinine ratio (mg/g) in urine.

2.5. Exclusion immune biomarkers

For chemokine detection, the Luminex-based protocol described by Muhlbacher, J et al (2020,Front Med,7:114) was used. To quantify chemokine (C-X-C motif) ligand (CXCL) 9 and CXCL 10, serum samples were conditioned to 10mM EDTA to prevent complement interference. Undiluted samples were measured in duplicate using a multiplex Human ProcartaPlex Simplex immunoassay (Thermo Fisher Scientific) according to the manufacturer's instructions. Immunoassays were performed on Luminex 200 instrument (Luminex corp.). Urine results were normalized to creatinine excretion and expressed in pg (chemokine)/mg (creatinine). Based on the detection of a defined set of single nucleotide polymorphisms detected by next generation sequencing on an Illumina MiSeq sequencer (Illumina Inc), dd-cfDNA levels in recipient plasma samples were detected using standard techniques, reflecting the extent of ongoing allograft damage.

2.6 monitoring of immune cells and leukocyte subpopulations

The underlying mechanisms of chronic antibody-mediated rejection, particularly the role of peripheral T cells and B cell subsets, have not been fully elucidated. Thus, prospective monitoring of immunophenotype under treatment with fevertuzumab is a promising approach to elucidate the impact on the immunomodulatory pathway when targeting CD 38. Furthermore, evaluation of plasma cell and NK cell counts allows monitoring of the pharmacodynamic effects of anti-CD 38 antibodies. To monitor leukocyte (sub) populations, phenotyping is performed using a reproducible immune monitoring panel (e.g., for flow cytometry). In the DuraClone kit, a predefined assay tube contains a layer with a series of ready-to-use dried (dry) antibody sets. Up to 10 different monoclonal antibodies per tube allow the recognition of sub-populations of leukocytes (e.g., T cells, B cells, NK cell sub-populations) present in whole blood samples.

To monitor immune cells, cells from blood, lymph nodes, bone marrow, spleen and grafts were stained using LIVE/DEADFixable Aqua Dead Cell Stain Kit (Life Technologies). Cells were then stained with one or more of the following mabs to human: CD3, CD4, CD8, CD14, CD20, CD25, CD27, CD28, CD38, CD56, CD95, CD127, CD159a, CD278 (ICOS), CD279 (PD-1), igM, igG, CXCR5 and (after fixation) Ki67 and FoxP3. Samples were collected by flow cytometry and analyzed for the percentage of cd38+ B cells and plasma cells, cd8+ T cells and/or cd4+, cd25+, CD 127-T cells using standard software (e.g., flowJo v 9.6.).

2.7 Gene expression analysis

For gene expression analysis, 5 mL blood was collected in PAXgene Blood RNA tubes and stored at-80 ℃ until retrospective analysis was performed. These tubes are designed to stabilize RNA in blood during long-term storage at ultra-low temperatures. Gene expression profiling (microarray analysis) was performed on peripheral blood to evaluate the effect of fevertuzumab on antibody-producing cells, and thus genes annotated as part of the B cell receptor signaling pathway were analyzed.

2.8 quantification of the Cyclovirus (TTV)

For TTV analysis, DNA was extracted from plasma samples using a NucliSENS easyMAG platform (biomeriux) and eluted in 50 μl of elution buffer. TTV DNA was quantified using TaqMan real-time PCR, for example, as described in Schiemann, M.et al (2017, transformation, 101:360-367). Quantitative PCR was performed using 2X TaqMan UniversalPCR Master Mix in a volume of 25. Mu.L (containing 5. Mu.L of extracted DNA, 400nM of each primer and 80nM probe). Using the CFX96 real-time system (Bio-Rad), the thermal cycle starts at 50 ℃ for 3 minutes, then at 95 ℃ for 10 minutes, then 45 cycles are performed as follows: 15 seconds at 95 ℃, 30 seconds at 55 ℃, and 30 seconds at 72 ℃. The results are reported as copy number/mL.

2.9 vaccine titers Process

Specific serum IgG titers of mumps, measles and rubella (MMR) were analyzed by standard ELISA techniques.

2.10 production of biological Material (out of routine monitoring)

Plasma (10 mL; chemokine, TTV loading), serum (10 mL; HLA antibody studies), whole blood (10 mL; RNA for gene expression analysis by flow cytometry) and urine (10 mL) (3X 30 mL peripheral blood) were collected before the study began (day 0), after 6 months and after 12 months. Finally, to measure the concentration of phenanthrenetuzumab and ADA, serum was obtained at each study visit (5 mL of peripheral blood per visit; a total of 18 visits).

Example 3: safety and efficacy of MOR202 in preventing and treating ABMR in non-human primates undergoing kidney transplantation

3.1 experiment NHP model

The present study was aimed at examining the safety and efficacy of MOR202 against desensitization (e.g., reduction of preformed antibodies), prevention of ABMR and ABMR after acute transplantation in a highly sensitized non-human primate kidney transplant model (see Kwun J. Et al. J Am Soc neprol. 2019 Jul;30 (7): 1206-1219). In addition, the long term effect of MOR202 on prevention of anti-elastic Donor Specific Antibodies (DSA) and advanced/chronic ABMR was assessed.

3.1.1 CD38 expression

CD38 expression levels on plasma cells from BM, spleen, lymph nodes and blood of recipient animals were analyzed for cross-reactivity with MOR 202. CD38 expression levels on red blood cells were examined to estimate the risk of anemia.

3.1.2 desensitization with MOR202

For allosensitization, male rhesus monkeys (Macaca mulatta) were sensitized to MHC mismatched donors by placing skin grafts at 8 week intervals twice in succession, as described in Burchuber CK et al Am J Transplay 19:724-736. Monkeys were treated with 16mg/kg of MOR202 for 4 weeks, about 8-12 weeks after the second skin graft. The level of alloantibodies was then measured. The level of desensitization was compared with the results of desensitization strategies using proteasome inhibitors (bortezomib/carfilzomib) for co-stimulatory blockade alone or in combination (Kwun J. Et al Blood adv. 2017 Nov 14; 1 (24): 2115-2119). CMV titers were measured before and after completion of drug treatment. To monitor immune cells, cells from blood, lymph nodes, bone marrow, spleen, and grafts were assessed by flow cytometry.

3.1.3 Curative effect of ABMR after desensitization treatment for MOR202 prevention and treatment

Animals received kidney transplants from the same skin transplant donor, which received MOR202 weekly for 4 weeks in addition to anti-rejection immunosuppression with rATG, tacrolimus, steroids. Kidney transplantation was performed essentially as described by Burchuber CK, et al (Am J Transplay. 2016;16 (6): 1726-1738). For depletion of plasma cell populations, sensitized rhesus monkeys were treated weekly with MOR 202. Control animals did not receive any treatment prior to kidney transplantation. Circulating B-cell and T-cell populations were assessed by FACS, as CD38 was expressed in both hematopoietic and non-hematopoietic cells (including activated B-cell and T-cell populations). These include circulating B cells, igg+b cells and memory B cells (igg+cd27+cd20+), as well as initial (native) (cd28+cd95-), central memory (cd28+cd95+) and effector memory (CD 28-cd95 int) subsets of CD4 and CD 8T cells.

Renal biopsy samples were collected at 1 month, 3 months, 6 months and at sacrifice, analyzed by (immune) histology, and scored according to Banff criteria. Donor-specific antibodies (DSA) after transplantation were measured weekly thereafter. Animals with rebound DSA that showed elevated serum creatinine were also treated with MOR202 for one month. Cells and humoral immune responses were analyzed, including follicular helper T cells, plasma cells (BM, LN and blood) and plasmablasts (blood and LN). Additional kidney graft biopsy samples were collected as needed. H & E, PAS and C4d staining was performed to monitor subclinical rejection and C4d deposition.

3.1.4 DSA monitoring

DSA levels were measured weekly continuously by flow cross-matching using donor lymphocytes and acceptor serum as described by Burghuber CK et al (Am J transfer 19:724-736). Briefly, donor PBMCs or spleen cells were incubated with recipient serum, washed, and stained with FITC-labeled anti-monkey IgG, anti-CD 20 mAb, and anti-CD 3 mAb. The Mean Fluorescence Intensity (MFI) of anti-monkey IgG on T cells or B cells was measured and expressed as MFI change from the pre-sensitization time point. NHP serum alloantibodies can also be measured using a human solid phase Luminex assay, which uses single HLA antigen beads (labscreen single antigen; one Lambda) to detect cross-reactive antibodies.

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Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Leu Pro Leu Val Tyr Thr Gly Phe Ala Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 8

<211> 109

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/note= "manual sequence description: synthetic polypeptide'

<400> 8

Asp Ile Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln

1 5 10 15

Thr Ala Arg Ile Ser Cys Ser Gly Asp Asn Leu Arg His Tyr Tyr Val

20 25 30

Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Gly Asp Ser Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Thr Tyr Thr Gly Gly Ala Ser Leu

85 90 95

Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln

100 105

<210> 9

<211> 300

<212> PRT

<213> person

<400> 9

Met Ala Asn Cys Glu Phe Ser Pro Val Ser Gly Asp Lys Pro Cys Cys

1 5 10 15

Arg Leu Ser Arg Arg Ala Gln Leu Cys Leu Gly Val Ser Ile Leu Val

20 25 30

Leu Ile Leu Val Val Val Leu Ala Val Val Val Pro Arg Trp Arg Gln

35 40 45

Gln Trp Ser Gly Pro Gly Thr Thr Lys Arg Phe Pro Glu Thr Val Leu

50 55 60

Ala Arg Cys Val Lys Tyr Thr Glu Ile His Pro Glu Met Arg His Val

65 70 75 80

Asp Cys Gln Ser Val Trp Asp Ala Phe Lys Gly Ala Phe Ile Ser Lys

85 90 95

His Pro Cys Asn Ile Thr Glu Glu Asp Tyr Gln Pro Leu Met Lys Leu

100 105 110

Gly Thr Gln Thr Val Pro Cys Asn Lys Ile Leu Leu Trp Ser Arg Ile

115 120 125

Lys Asp Leu Ala His Gln Phe Thr Gln Val Gln Arg Asp Met Phe Thr

130 135 140

Leu Glu Asp Thr Leu Leu Gly Tyr Leu Ala Asp Asp Leu Thr Trp Cys

145 150 155 160

Gly Glu Phe Asn Thr Ser Lys Ile Asn Tyr Gln Ser Cys Pro Asp Trp

165 170 175

Arg Lys Asp Cys Ser Asn Asn Pro Val Ser Val Phe Trp Lys Thr Val

180 185 190

Ser Arg Arg Phe Ala Glu Ala Ala Cys Asp Val Val His Val Met Leu

195 200 205

Asn Gly Ser Arg Ser Lys Ile Phe Asp Lys Asn Ser Thr Phe Gly Ser

210 215 220

Val Glu Val His Asn Leu Gln Pro Glu Lys Val Gln Thr Leu Glu Ala

225 230 235 240

Trp Val Ile His Gly Gly Arg Glu Asp Ser Arg Asp Leu Cys Gln Asp

245 250 255

Pro Thr Ile Lys Glu Leu Glu Ser Ile Ile Ser Lys Arg Asn Ile Gln

260 265 270

Phe Ser Cys Lys Asn Ile Tyr Arg Pro Asp Lys Phe Leu Gln Cys Val

275 280 285

Lys Asn Pro Glu Asp Ser Ser Cys Thr Ser Glu Ile

290 295 300

<210> 10

<211> 360

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/note= "manual sequence description: synthetic Polynucleotide'

<400> 10

caggtgcaat tggtggaaag cggcggcggc ctggtgcaac cgggcggcag cctgcgtctg 60

agctgcgcgg cctccggatt taccttttct tcttattata tgaattgggt gcgccaagcc 120

cctgggaagg gtctcgagtg ggtgagcggt atctctggtg atcctagcaa tacctattat 180

gcggatagcg tgaaaggccg ttttaccatt tcacgtgata attcgaaaaa caccctgtat 240

ctgcaaatga acagcctgcg tgcggaagat acggccgtgt attattgcgc gcgtgatctt 300

cctcttgttt atactggttt tgcttattgg ggccaaggca ccctggtgac ggttagctca 360

<210> 11

<211> 327

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/note= "manual sequence description: synthetic Polynucleotide'

<400> 11

gatatcgaac tgacccagcc gccttcagtg agcgttgcac caggtcagac cgcgcgtatc 60

tcgtgtagcg gcgataatct tcgtcattat tatgtttatt ggtaccagca gaaacccggg 120

caggcgccag ttcttgtgat ttatggtgat tctaagcgtc cctcaggcat cccggaacgc 180

tttagcggat ccaacagcgg caacaccgcg accctgacca ttagcggcac tcaggcggaa 240

gacgaagcgg attattattg ccagacttat actggtggtg cttctcttgt gtttggcggc 300

ggcacgaagt taaccgttct tggccag 327

Claims (15)

1. An anti-CD 38 antibody or antibody fragment for use in the treatment and/or prevention of organ transplant antibody-mediated rejection in a human subject.

2. An anti-CD 38 antibody or antibody fragment for use according to claim 1, wherein

The organ transplant is a kidney, heart, liver, lung, pancreas, stomach, skin or intestine transplant.

3. An anti-CD 38 antibody or antibody fragment for use according to claim 1 or 2, wherein

The antibodies comprise an HCDR1 region of amino acid sequence SEQ ID NO. 1, an HCDR2 region of amino acid sequence SEQ ID NO. 2, an HCDR3 region of amino acid sequence SEQ ID NO. 3, an LCDR1 region of amino acid sequence SEQ ID NO. 4, an LCDR2 of amino acid sequence SEQ ID NO. 5, and an LCDR3 region of amino acid sequence SEQ ID NO. 6.

4. An anti-CD 38 antibody or antibody fragment for use according to claim 3, wherein

The anti-CD 38 antibody or antibody fragment comprises a variable heavy chain (VH) region of SEQ ID No. 7 and a variable light chain (VL) region of SEQ ID No. 8.

5. An anti-CD 38 antibody or antibody fragment for use according to any one of the preceding claims, wherein

The antibody or antibody fragment specific for CD38 is IgG1.

6. An anti-CD 38 antibody or antibody fragment for use according to any one of the preceding claims, wherein

The antibody or antibody fragment specific for CD38 is a human antibody.

7. An anti-CD 38 antibody or antibody fragment for use according to any one of the preceding claims, wherein

The antibody or antibody fragment specific for CD38 is fezetuzumab.

8. An anti-CD 38 antibody or antibody fragment for use according to any one of the preceding claims, wherein

The antibodies deplete plasma cells by ADCC and/or ADCP.

9. An anti-CD 38 antibody or antibody fragment for use according to any one of the preceding claims, wherein

Administration of the anti-CD 38 antibody or antibody fragment results in a reduction of cd38+ antibody secreting cells.

10. An anti-CD 38 antibody or antibody fragment for use according to any one of the preceding claims, wherein

Administration of the anti-CD 38 antibody or antibody fragment results in a decrease in anti-HLA antibody levels.

11. An anti-CD 38 antibody or antibody fragment for use according to claim 10, wherein

Administration of the anti-CD 38 antibody or antibody fragment results in reduced levels of class I and/or class II anti-HLA antibodies.

12. An anti-CD 38 antibody or antibody fragment for use according to claim 11, wherein

Administration of the anti-CD 38 antibody or antibody fragment results in a decrease in anti-DQ 5 antibody levels.

13. The anti-CD 38 antibody or antibody fragment for use according to any one of the preceding claims, wherein the antibody or antibody fragment is administered at 16mg/kgi.v.

14. An anti-CD 38 antibody or antibody fragment for use according to claim 13, wherein

At least 2, at least 5, at least 7, or at least 9 doses of the antibody or antibody fragment are dosed.

15. An anti-CD 38 antibody or antibody fragment for use according to any one of the preceding claims, wherein

The subjects to be treated are characterized in that eGFR.gtoreq.20 ml/min/1.73m according to the CKD-EPI equation ² 。