CD31 competitors and uses thereof

文档序号：1803581 发布日期：2021-11-05 浏览：17次中文

阅读说明：本技术 Cd31竞争剂及其用途 (CD31 competitors and uses thereof ) 是由 L·布雷萨克 S·格雷罗 D·图洛格于 2020-01-23 设计创作，主要内容包括：本发明涉及分离的抗人CD38抗体或其抗原结合片段；编码其的核酸和表达载体。本发明进一步涉及与CD31特异性竞争结合CD38的化合物,用于预防和/或治疗有此需要的受试者中的疾病,所述疾病选自神经变性疾病、神经炎性疾病、炎性疾病、自身免疫疾病、代谢性疾病、眼部疾病、年龄相关疾病、癌症和转移。(The present invention relates to isolated anti-human CD38 antibodies or antigen-binding fragments thereof; nucleic acids encoding the same and expression vectors. The present invention further relates to compounds that specifically compete with CD31 for binding to CD38 for use in the prevention and/or treatment of a disease selected from the group consisting of neurodegenerative diseases, neuroinflammatory diseases, inflammatory diseases, autoimmune diseases, metabolic diseases, ocular diseases, age-related diseases, cancer and metastasis in a subject in need thereof.)

1. An isolated anti-human CD38 antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof competes with CD31, preferably with human CD 31; and induces lysosomal exocytosis in the cell, which is inhibited in the presence of vacuolin-1.

2. The isolated anti-human CD38 antibody or antigen-binding fragment thereof of claim 1, wherein the isolated anti-human CD38 antibody or antigen-binding fragment thereof is monoclonal.

3. The isolated anti-human CD38 antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the isolated anti-human CD38 antibody or antigen-binding fragment thereof is humanized.

4. The isolated anti-human CD38 antibody or antigen-binding fragment thereof of any one of claims 1-3, wherein:

a) the Heavy Chain Variable Region (HCVR) of the isolated anti-human CD38 antibody or antigen-binding fragment thereof comprises the following three Complementarity Determining Regions (CDRs):

-V_H-CDR1：GFTFSNX₁(SEQ ID NO:4)，

-V_H-CDR2：X₂GSSRX₃(SEQ ID NO:5), and

-V_H-CDR3：X₄X₅X₆X₇X₈YX₉X₁₀X₁₁X₁₂GMDV (SEQ ID NO: 6); and is

b) The Light Chain Variable Region (LCVR) of the isolated anti-human CD38 antibody or antigen-binding fragment thereof comprises the following three CDRs:

-V_L-CDR1：AGTSSDVGGX₁₃X₁₄X₁₅VS(SEQ ID NO:21)，

-V_L-CDR2：X₁₆DSX₁₇RPS (SEQ ID NO:22), and

-V_L-CDR3：STRVFGGGT(SEQ ID NO:23)；

wherein:

X₁selected from Tyr (Y), Asn (N), and Ser (S);

X₂selected from Ser (S) and Tyr (Y);

X₃selected from Tyr (Y), Asp (D), Asn (N) and Ser (S);

X₄selected from ser(s) and a vacancy;

X₅selected from ser(s) and a vacancy;

X₆selected from Ser (S) and Tyr (Y);

X₇selected from Ser (S) and Asp (D);

X₈selected from Tyr (Y), Ser (S), Asp (D) and Gly (G);

X₉selected from Tyr (Y) and Gly (G);

X₁₀selected from Ser (S), Tyr (Y) and Phe (F);

X₁₁selected from Gly (G) and Asp (D);

X₁₂selected from Asn (N), Tyr (Y) and Ser (S);

X₁₃selected from Ser (S) and Asn (N);

X₁₄selected from Ser (S) and Tyr (Y);

X₁₅selected from Tyr (Y) and Ser (S);

X₁₆selected from Tyr (Y), Ser (S), and Asp (D); and is

X₁₇Selected from Tyr (Y) and Asn (N).

5. The isolated anti-human CD38 antibody or antigen-binding fragment thereof of any one of claims 1-4, wherein the anti-human CD38 antibody or antigen-binding fragment thereof comprises:

-sequence SEQ ID NO: v of 7_H-CDR1, sequence SEQ ID NO: v of 10_H-CDR2, sequence SEQ ID NO: v of 15_H-CDR3, sequence SEQ ID NO: v of 24_L-CDR1, sequence SEQ ID NO: v of 29_LCDR2 and V having the sequence SEQ ID NO. 23_L-CDR3；

-sequence SEQ ID NO: v of 8_H-CDR1, sequence SEQ ID NO: v of 11_H-CDR2, sequence SEQ ID NO: v of 16_H-CDR3, sequence SEQ ID NO: v of 25_L-CDR1, sequence SEQ ID NO: v of 30_LCDR2 and V having the sequence SEQ ID NO. 23_L-CDR3；

-sequence SEQ ID NO: v of 9_H-CDR1, sequence SEQ ID NO: 12V_H-CDR2, sequence SEQ ID NO: v of 17_H-CDR3, sequence SEQ ID NO: v of 25_L-CDR1, sequence SEQ ID NO: v of 30_LCDR2 and V having the sequence SEQ ID NO. 23_L-CDR3；

-sequence SEQ ID NO: v of 7_H-CDR1, sequence SEQ ID NO: v of 13_H-CDR2, sequence SEQ ID NO: v of 19_H-CDR3, sequence SEQ ID NO: v of 27_L-CDR1, sequence SEQ ID NO: v of 32_LCDR2 and V having the sequence SEQ ID NO. 23_L-CDR3；

-sequence is SEQID NO: v of 9_H-CDR1, sequence SEQ ID NO: v of 14_H-CDR2, sequence SEQ ID NO: v of 20_H-CDR3, sequence SEQ ID NO: v of 28_L-CDR1, sequence SEQ ID NO: v of 30_LCDR2 and V having the sequence SEQ ID NO. 23_L-a CDR 3; or

-sequence SEQ ID NO: v of 9_H-CDR1, sequence SEQ ID NO: v of 14_H-CDR2, sequence SEQ ID NO: v of 16_H-CDR3, sequence SEQ ID NO: v of 28_L-CDR1, sequence SEQ ID NO: v of 30_LCDR2 and V having the sequence SEQ ID NO. 23_L-CDR3。

6. The isolated anti-human CD38 antibody or antigen-binding fragment thereof of any one of claims 1-5, wherein the anti-human CD38 antibody or antigen-binding fragment thereof comprises:

-a HCVR of sequence SEQ ID No. 33 and a LCVR of sequence SEQ ID No. 39;

-a HCVR of sequence SEQ ID No. 34 and a LCVR of sequence SEQ ID No. 40;

-a HCVR of sequence SEQ ID No. 35 and a LCVR of sequence SEQ ID No. 40;

-a HCVR of sequence SEQ ID No. 36 and a LCVR of sequence SEQ ID No. 41;

-a HCVR of sequence SEQ ID No. 37 and a LCVR of sequence SEQ ID No. 42; or

-a HCVR of sequence SEQ ID No. 38 and a LCVR of sequence SEQ ID No. 42.

7. A nucleic acid encoding the isolated anti-human CD38 antibody or antigen-binding fragment thereof of any one of claims 1-6.

8. An expression vector comprising the nucleic acid of claim 7.

9. A compound that specifically competes for binding to CD38 with CD31 for use in the prevention and/or treatment of a disease selected from the group consisting of a neurodegenerative disease; a neuroinflammatory disorder; inflammatory diseases; autoimmune diseases; metabolic diseases; eye diseases; age-related diseases; and cancer and metastasis.

10. The compound of claim 9, wherein the disease is selected from amyotrophic lateral sclerosis; parkinson's disease and related disorders; alzheimer's disease and related disorders; huntington's disease; multiple sclerosis; rheumatoid arthritis; systemic lupus erythematosus; diabetes mellitus; obesity; non-alcoholic steatohepatitis; age-related macular degeneration; and glaucoma.

11. A compound that specifically competes for binding to CD38 with CD31 for use in increasing the level of at least one anti-inflammatory cytokine in a subject in need thereof, particularly in the blood of the subject.

12. The compound of claim 11, wherein the anti-inflammatory cytokine is interleukin-10 (IL-10).

13. The compound of any one of claims 9 to 12, wherein the compound is selected from the group consisting of a peptide, a chimeric peptide, an antibody, an antigen-binding fragment thereof, an antigen-binding antibody mimetic, an oligonucleotide, and a small organic molecule; and wherein the compound:

-inducing tyrosine phosphorylation of discrete cytoplasmic matrices in the cell, which is inhibited in the presence of genistein; and/or

-inducing lysosomal exocytosis in the cell, which is inhibited in the presence of vacuolin-1.

14. The compound of any one of claims 9-13, wherein the compound is the isolated anti-human CD38 antibody or antigen-binding fragment thereof of any one of claims 1-6.

15. The compound of any one of claims 9 to 13, wherein the compound is:

-a peptide comprising Ig-like domains 1-3 of human CD31, preferably a peptide comprising amino acids 28 to 315 of SEQ ID No. 1 or a variant thereof; or

-a chimeric peptide comprising Ig-like domains 1-3 of human CD31, preferably comprising the amino acid sequence of SEQ ID NO:1 or a variant thereof; and to the Fc domain of immunoglobulins, to human serum albumin, preferably to domain III of human serum albumin or to transferrin.

Technical Field

The present invention relates to the field of prevention and/or treatment of diseases.

In particular, the present invention relates to compounds which are competitive agents (competitors) for CD31 for use in the prevention and/or treatment of disease.

Background

CD31, also known as Platelet endothelial cell adhesion molecule-1 (PECAM-1), is a 130kDa type I transmembrane glycoprotein consisting of a 6 extracellular immunoglobulin (Ig) -like homeodomain, a 19-residue transmembrane domain and a 118-residue cytoplasmic tail (Newman and Newman,2003.Arterioscler Thromb Vasc biol.23: 953-. Thus, CD31 belongs to the Ig superfamily of cell adhesion molecules. CD31 is capable of binding to itself through a homophilic antigen interaction (homographic interaction) involving Ig-like domain 1; and binding to two other ligands α V β 3 integrin and CD38 through xenotropic antigen interactions involving Ig-like domains 1-3 (Newman,1997.J Clin invest.99(1): 3-8).

CD31 expression is observed primarily in endothelial cells, where it is considered a constitutive marker (Kalinowska & Losy,2006.Eur J neurol.13(12):1284-90), but CD31 expression is also observed in most non-erythroid cell subsets of the hematopoietic lineage (hematopoietic linkage), including platelets, monocytes, neutrophils, T and B cells (Wang et al, 2003.Am J Physiol Heart Circuit physiol.284(3): H1008-17). Although CD31 alloantigen interaction is a major component of endothelial Cell-Cell junctions, it is also involved in the process of leukocyte-endothelial Cell migration (diapedesis), which allows leukocytes to penetrate into tissues during inflammation (Ilan & Madri,2003.Curr Opin Cell biol.15(5): 515-24).

CD31 was found to exist in membrane and soluble forms following metalloprotease dependent cleavage (Ilan et al, 2001.FASEB J.15(2): 362-72). Increased circulating levels of soluble CD31(sCD31) have been observed in some inflammatory diseases including atherosclerosis and sepsis (Feng et al, 2016.Eur Rev Med Pharmacol Sci.20(19): 4082-. Interestingly, increased levels of cerebrospinal fluid (CSF) sCD31 were also observed in a variety of neurodegenerative diseases including multiple sclerosis (Losy et al, 1999.J Neurommunol.99 (2):169-72, Kuenz et al, 2005.J Neurommunol.167 (1-2):143-9), HIV-encephalitis (Eugenin et al, 2006.J Leukoc biol.79(3):444-52), paraneoplastic encephalo-myelo-polyneuropathy (paraneoplastic encephalo-myo-polyneuropathopathy) (Dziewska et al, 2000.Folia Neurophothol.38 (1):29-33) and cerebral ischemia (Zaremba and Losy,2002.Acta Neurol Scan.106 (5): 292-8). A common understanding of these increased brain sCD31 levels is that they indicate an impaired Blood Brain Barrier (BBB), a characteristic feature of any neuroinflammatory response (Kalinowska and Losy,2006.Eur J neurol.13(12): 1284-90).

However, the level of brain sCD31 following ischemic stroke was found to be positively correlated with (i) the severity of neurological stroke and (ii) the degree of dysfunction (Zaremba and Losy,2002.Central-European J immunol.27: 90-96). Thus, applicants wondered whether sCD31 was actively involved in the neurodegenerative process. Indeed, sCD31 itself may also be toxic to neurons. Unexpectedly, applicants have found that sCD31 is not toxic, but in contrast, it has a strong protective effect on neurons in vitro. This result is particularly surprising, since CD31 expression in the brain is restricted to endothelial cells of the BBB (Williams et al, 1996.J Neurosci Res.45(6): 747-57). The neuroprotective effect of sCD31 was antagonized in the presence of neutralizing clone Moon-1 anti-CD 31 antibody and by antagonist anti-CD 38 antibody (clone OKT10 or AT13/5), indicating that the neuroprotective effect of sCD31 is mediated by interaction with its ligand CD 38. Furthermore, the neuroprotective effect of sCD31 was recapitulated by an agonistic anti-CD 38 antibody, which was previously shown to mimic the interaction between sCD31 and CD38 (Deaglio et al, 1998.J Immunol.160(1): 395-402). Notably, the neuroprotective effect of the agonistic anti-CD 38 antibody (clone HB7) was inhibited in the presence of antagonistic anti-CD 38 antibodies (clone OKT10 or AT13/5), as was the neuroprotective effect of sCD 31.

CD38 is a 45kDa type II transmembrane glycoprotein with a long C-terminal extracellular domain and a short N-terminal cytoplasmic domain. CD38 has a variety of biological activities, including receptor-mediated functions (via internalization), tyrosine phosphorylation-mediated functions, and enzyme-mediated functions. Furthermore, as disclosed in WO2018224683, some specific anti-CD 38 antibodies were reported to be of interest in cancer treatment. Applicants herein found that the neuroprotective effects of sCD31 or an agonistic anti-CD 38 antibody (clone HB7) were antagonized when tyrosine phosphorylation was inhibited.

Disclosure of Invention

The present invention relates to an isolated anti-human CD38 antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof competes with CD31, preferably with human CD 31; and induces lysosomal exocytosis in the cell, which is inhibited in the presence of vacuolin-1.

In one embodiment, the isolated anti-human CD38 antibody or antigen-binding fragment thereof is monoclonal.

In one embodiment, the isolated anti-human CD38 antibody or antigen-binding fragment thereof is humanized.

In one embodiment of the process of the present invention,

a) the Heavy Chain Variable Region (HCVR) of the isolated anti-human CD38 antibody or antigen-binding fragment thereof comprises the following three Complementarity Determining Regions (CDRs):

-V_H-CDR1：GFTFSNX₁(SEQ ID NO:4)，

-V_H-CDR2：X₂GSSRX₃(SEQ ID NO:5), and

-V_H-CDR3：X₄X₅X₆X₇X₈YX₉X₁₀X₁₁X₁₂GMDV (SEQ ID NO: 6); and

b) the Light Chain Variable Region (LCVR) of the isolated anti-human CD38 antibody or antigen-binding fragment thereof comprises the following three CDRs:

-V_L-CDR1：AGTSSDVGGX₁₃X₁₄X₁₅VS(SEQ ID NO:21)，

-V_L-CDR2：X₁₆DSX₁₇RPS (SEQ ID NO:22), and

-V_L-CDR3：STRVFGGGT(SEQ ID NO:23)；

wherein:

X₁selected from Tyr (Y), Asn (N), and Ser (S);

X₂selected from Ser (S) and Tyr (Y);

X₃selected from Tyr (Y), Asp (D), Asn (N) and Ser (S);

X₄selected from ser(s) and null position (empty position);

X₅selected from ser(s) and a vacancy;

X₆selected from Ser (S) and Tyr (Y);

X₇selected from Ser (S) and Asp (D);

X₈selected from Tyr (Y), Ser (S), Asp (D) and Gly (G);

X₉selected from Tyr (Y) and Gly (G);

X₁₀selected from Ser (S), Tyr (Y) and Phe (F);

X₁₁selected from Gly (G) and Asp (D);

X₁₂selected from Asn (N), Tyr (Y) and Ser (S);

X₁₃selected from Ser (S) and Asn (N);

X₁₄selected from Ser (S) and Tyr (Y);

X₁₅selected from Tyr (Y) and Ser (S);

X₁₆selected from Tyr (Y), Ser (S), and Asp (D); and is

X₁₇Selected from Tyr (Y) and Asn (N).

In one embodiment, the anti-human CD38 antibody or antigen-binding fragment thereof comprises:

-sequence SEQ ID NO: v of 9_H-CDR1, sequence SEQ ID NO: v of 14_H-CDR2, sequence SEQ ID NO: v of 20_H-CDR3, sequence SEQ ID NO: v of 28_L-CDR1, sequence SEQ ID NO: v of 30_LCDR2 and V having the sequence SEQ ID NO. 23_L-a CDR 3; or

In one embodiment, the anti-human CD38 antibody or antigen-binding fragment thereof comprises:

-a HCVR of sequence SEQ ID No. 33 and a LCVR of sequence SEQ ID No. 39;

-a HCVR of sequence SEQ ID No. 34 and a LCVR of sequence SEQ ID No. 40;

-a HCVR of sequence SEQ ID No. 35 and a LCVR of sequence SEQ ID No. 40;

-a HCVR of sequence SEQ ID No. 36 and a LCVR of sequence SEQ ID No. 41;

-a HCVR of sequence SEQ ID No. 37 and a LCVR of sequence SEQ ID No. 42; or

-a HCVR of sequence SEQ ID No. 38 and a LCVR of sequence SEQ ID No. 42.

The invention also relates to nucleic acids encoding the isolated anti-human CD38 antibodies or antigen-binding fragments thereof described herein.

The invention also relates to expression vectors comprising the nucleic acids of the invention.

The present invention also relates to a compound that specifically competes for binding to CD38 with CD31 for use in the prevention and/or treatment of a disease selected from the group consisting of neurodegenerative diseases; a neuroinflammatory disorder; inflammatory diseases; autoimmune diseases; metabolic diseases; eye diseases; age-related diseases; and cancer and metastasis.

In one embodiment, the disease is selected from amyotrophic lateral sclerosis; parkinson's disease and related disorders; alzheimer's disease and related disorders; huntington's disease; multiple sclerosis; rheumatoid arthritis; systemic lupus erythematosus; diabetes mellitus; obesity; non-alcoholic steatohepatitis; age-related macular degeneration; and glaucoma.

The present invention also relates to compounds that specifically compete with CD31 for binding to CD38, for use in increasing the level of at least one anti-inflammatory cytokine in the blood of a subject in need thereof, in particular said subject.

In one embodiment, the anti-inflammatory cytokine is interleukin-10 (IL-10).

In one embodiment, the compound used in any of the methods of the invention is selected from the group consisting of a peptide, a chimeric peptide, an antibody, an antigen-binding fragment thereof, an antigen-binding antibody mimetic, an oligonucleotide, and a small organic molecule; and the compound:

-inducing tyrosine phosphorylation of discrete cytoplasmic matrices in the cell, which is inhibited in the presence of genistein; and/or

-inducing lysosomal exocytosis in the cell, which is inhibited in the presence of vacuolin-1.

In one embodiment, the compound used in any of the methods of the invention is an isolated anti-human CD38 antibody or antigen-binding fragment thereof described herein.

In one embodiment, the compound used in any of the methods of the invention is:

-a peptide comprising Ig-like domains 1-3 of human CD31, preferably a peptide comprising amino acids 28 to 315 of SEQ ID No. 1 or a variant thereof; or

-a chimeric peptide comprising Ig-like domains 1-3 of human CD31, preferably comprising the amino acid sequence of SEQ ID NO:1 or a variant thereof; fused to the Fc domain of an immunoglobulin, to human serum albumin, preferably to domain III of human serum albumin or to transferrin.

Definition of

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the relevant art. The meanings of certain terms and phrases used in the specification, examples, and claims are provided for convenience.

As used herein, "Affitin" refers to an artificial protein having the ability to selectively bind to an antigen. They are structurally derived from the DNA-binding protein Sac7d, which is found in the microorganism Sulfolobus acidocaldarius (Sulfolobus acidocaldarius) belonging to the archaea domain. By randomizing the amino acids on the Sac7d binding surface, e.g. by generating randomly substituted variants corresponding to 11 residues of the Sac7d binding interface, it is possible to generate libraries of affitin, which can be directed against multiple targets, e.g. peptides, proteins, viruses and bacteria, and to subject the resulting protein libraries to multiple rounds of ribosome display. Affitin is an antibody mimetic and is being developed as a tool in biotechnology. They are also useful as specific inhibitors of various enzymes (Krehenbrink et al, 2008.J Mol biol.383(5): 1058-68). The skilled person can easily develop affitin with the desired binding properties using methods known in the art, in particular as disclosed in international patent application WO2008068637 and the publications cited above, in particular to generate phage display and/or ribosome display libraries and to screen them using the antigens disclosed herein.

As used herein, "antibody" or "immunoglobulin" refers to a protein having a combination of two heavy chains and two light chains, whether or not it has any associated specific immunoreactivity. By "antibody" is meant such an assembly (assembly) having significant known specific immunoreactivity to an antigen of interest. The term encompasses polyclonal antibodies, monoclonal antibodies, recombinant antibodies, bispecific antibodies, multispecific antibodies, and modified antibodies.

It is also understood that, as used herein, the terms "antibody" and "immunoglobulin" encompass an antibody or antibody binding fragment that has been modified using known methods. For example, to slow clearance (clearance) in vivo and achieve more desirable pharmacokinetic profiles, polyethylene glycol (PEG) may be used to modify the antibody or binding fragment thereof. Methods for coupling (conjugating) and site-specific conjugation of PEG to antibodies or binding fragments thereof are described, for example, in Leong et al, 2001.cytokine.16(3): 106-19; delgado et al, 1996, Br J cancer.73(2): 175-82.

Antibodies and immunoglobulins comprise a light chain and a heavy chain with or without an interchain covalent bond between them. The basic immunoglobulin structure in vertebrate systems is relatively well understood. The general term "immunoglobulin" includes five different classes of antibodies that can be biochemically distinguished. Although the following discussion is directed generally to the IgG class of immunoglobulin molecules, all five antibody classes are within the scope of the invention. For IgG, the immunoglobulin comprises two identical light polypeptide chains of about 23kDa molecular weight and two identical heavy chains of about 53-70kDa molecular weight. These four chains are linked in a "Y" configuration by disulfide bonds, where the light chain begins at the "Y" mouth and continues to embrace the heavy chain through the variable region (blacket). The light chain of antibodies is classified as kappa (. kappa.) or lambda (. lamda.). Each heavy chain class may be associated with a kappa or lambda light chain. Typically, when the immunoglobulin is produced by a hybridoma, B cell, or genetically engineered host cell, the light and heavy chains are covalently bonded to each other, and the "tail" regions of the two heavy chains are bonded to each other by covalent disulfide bonds or non-covalent bonds. In the heavy chain, the amino acid sequence extends from the N-terminus of the Y-configuration divergent ends to the C-terminus at the bottom of each chainAnd (4) an end. Those skilled in the art will appreciate that heavy chains are classified as gamma (γ), mu (μ), alpha (α), delta (δ), or epsilon (ε), with some subclasses (e.g., γ 1- γ 4) in between. It is the nature of this chain that determines the "class" of antibodies to be IgG, IgM, IgA, IgD or IgE, respectively. The immunoglobulin subclasses or "isotypes" (e.g., IgGl, IgG2, IgG3, IgG4, IgA1, etc.) have been well characterized and are known to confer functional specificity. Modified forms of each of these classes and isoforms are readily discernible to those of skill in the art in view of this disclosure and are therefore within the scope of the invention. As described above, the variable region of an antibody allows the antibody to selectively recognize and specifically bind to an epitope on an antigen. That is, the light chain variable domain (V) of the antibody_LDomains) and heavy chain variable domains (V)_HDomains) to form variable regions that define a three-dimensional antigen binding site. This quaternary antibody structure forms the antigen binding site present at the end of each arm of the "Y". More specifically, the antigen binding site consists of V_HAnd V_LThree Complementarity Determining Regions (CDRs) on each of the domains are defined.

Various studies to develop therapeutic antibodies have engineered the Fc region to optimize antibody properties to produce molecules more tailored to their desired pharmacological activity. The Fc region of an antibody mediates its serum half-life and effector functions such as Complement Dependent Cytotoxicity (CDC), Antibody Dependent Cellular Cytotoxicity (ADCC), and Antibody Dependent Cellular Phagocytosis (ADCP). Several mutations at the interface between CH2 and CH3 domains, such as N297A, N297G, L234A/L235A, L235E, T250Q/M428L, M252Y/S254T/T256E and H433K/N434F, have been shown to increase the binding affinity for FcRn and the half-life of IgG1 in vivo. However, there is not always a direct relationship between increased FcRn binding and increased half-life. One way to increase the efficacy of therapeutic antibodies (efficacy) is to increase their serum persistence (serum persistence), thereby allowing higher circulating levels, less frequent administration and reduced dosage. It may be desirable to engineer the Fc region to reduce or increase the effector function of the antibody. For antibodies that target cell surface molecules, especially antibodies on immune cells or neurons, effector functions need to be abrogated. Conversely, for antibodies intended for oncology, increasing effector function may improve therapeutic activity. The four human IgG isotypes bind with different affinities to the activating Fc γ receptor (Fc γ RI, Fc γ RIIa, Fc γ RIIIa), the inhibitory Fc γ RIIb receptor, and the first component of complement (C1q), resulting in very different effector functions. IgG binding to Fc γ R or C1q depends on residues located in the hinge region and the CH2 domain. Two regions of the CH2 domain are critical for Fc γ R and C1q binding and have unique sequences in IgG2 and IgG 4.

As used herein, "antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted antibodies bind to Fc receptors (fcrs) present on certain cytotoxic cells (e.g., NK cells, neutrophils, monocytes, and macrophages) enabling these cytotoxic effector cells to specifically bind to and subsequently kill target cells bearing antigens. To assess ADCC activity of a molecule of interest, an in vitro ADCC assay may be performed, such as those described in patents US5,500,362 or US5,821,337.

As used herein, "antibody-dependent phagocytosis" or "opsonization" refers to a cell-mediated reaction in which non-specific cytotoxic cells expressing Fc γ R recognize bound antibodies on target cells and subsequently cause phagocytosis of the target cells.

As used herein, "Anticalin" refers to an artificial protein capable of binding to an antigen (whether a protein or small molecule). They are antibody mimetics derived from the natural binding protein family of human lipocalins (lipocalins). Anticalin is about 8-fold smaller, with a size of about 180 amino acids and a mass of about 20kDa (Kolmar & Skerra,2008.FEBS J.275(11): 2667). An Anticalin phage display library has been generated that allows screening and selection, particularly anticalins with specific binding properties. The skilled person can readily develop anticalins with the desired binding properties using methods known in the art, in particular as disclosed in EP1270725, US patent US8,536,307, Schlehuber & Skerra (2002.Biophys chem.96(2-3):213-28) and the references cited above, in particular using the antigens described herein to generate and screen phage display and/or ribosome display libraries.

As used herein, "antigen-binding antibody mimetic" refers to artificial proteins, peptides, nucleic acids, and, more broadly, any chemical compound that has the ability to mimic an antibody's binding to an antigen.

Such mimetics include oligonucleotide aptamers, as well as affitin, anticalin, and peptide aptamers. affitin, anticalin and peptide aptamers can be produced in a variety of expression systems including bacterial expression systems or by combinatorial chemistry. Thus, the present invention provides affitin, anticalin, peptide aptamers, and other similar antigen-binding antibody mimetics (e.g., oligonucleotide aptamers) that have the characteristics of the antibodies described herein, particularly with respect to competition with CD 31.

As used herein, "antigen-binding fragment of an antibody" refers to a portion or region of an antibody that comprises fewer amino acid residues than an intact antibody, i.e., a molecule that corresponds to a portion of the structure of an antibody of the invention that exhibits antigen-binding ability to CD38, possibly in its native form; such fragments in particular exhibit the same or substantially the same antigen binding specificity for the antigen as compared to the antigen binding specificity of the corresponding intact antibody. Advantageously, the antigen binding fragment has a similar binding affinity as the corresponding intact antibody. However, antigen binding fragments having reduced antigen binding affinity relative to the corresponding intact antibody are also encompassed within the invention. Antigen binding capacity can be determined by measuring the affinity between the antibody and the target fragment. These antigen binding fragments may also be referred to as "functional fragments" of antibodies.

Antigen-binding fragments of antibodies include, but are not limited to, Fv, dsFv, scFv, Fab ', F (ab')2, and single domain antibodies. Fv fragments consisting of V of antibodies held together by hydrophobic interactions_LAnd V_HDomain composition; in the dsFv fragment, V_H:V_LHeterodimers are stabilized by disulfide bonds; in the scFv fragment, V_LAnd V_HThe domains are linked to each other by flexible peptide linkers, thereby forming single chain proteins. Fab fragments are monomeric fragments that can be obtained by papain digestion of antibodies; they contain the entire L chain and the V of the H chain_H-C_H1, joined together by disulfide bonds. F (ab')2 fragments can be generated by pepsin digestion of the antibody below the hinge disulfide bond; it comprises two Fab' fragments, and additionally a portion of the hinge region of an immunoglobulin molecule. Fab 'fragments can be obtained by cleavage of the disulfide bond in the hinge region of the F (ab')2 fragment. F (ab')2 fragments are bivalent, i.e., they contain two antigen binding sites, as do natural immunoglobulin molecules; in another aspect, Fv (V constituting the variable part of Fab)_H-V_LDimers), dsFv, scFv, Fab and Fab' fragments are monovalent, i.e., they comprise a single antigen binding site. These basic antigen-binding fragments of the invention may be combined together to obtain multivalent antigen-binding fragments, such as diabodies, triabodies or tetrabodies. These multivalent antigen binding fragments are also part of the present invention.

As used herein, "bispecific antibody" refers to an antibody that recognizes two different antigens due to having at least one region specific for a first antigen (e.g., derived from the variable region of a first antibody) and at least one second region specific for a second antigen (e.g., derived from the variable region of a second antibody). Bispecific antibodies specifically bind to two target antigens and are thus a multispecific antibody. Multispecific antibodies that recognize two or more different antigens may be produced by recombinant DNA methods or include, but are not limited to, antibodies chemically produced by any convenient method. Bispecific antibodies include all antibodies or antibody conjugates or polymeric forms of antibodies that are capable of recognizing two different antigens. Bispecific antibodies include antibodies that have been reduced and recombined to retain their bivalent character, as well as antibodies that have been chemically conjugated so that they may have multiple antigen recognition sites for each antigen, e.g., BiME (Bispecific Macrophage Enhancing antibody), BiTE (Bispecific T cell engager), DART (Dual affinity redirecting molecule); DNL (dock-and-lock), DVD-Ig (dual variable domain immunoglobulin), HAS (human serum albumin), kih (knobs into holes).

As used herein, "CD 31" refers to a 130kDa type I transmembrane glycoprotein, also known as platelet endothelial cell adhesion molecule-1 (PECAM-1), PECA1, GPIIA', EndocAM or CD31/Endocamas, and is described in Newman & Newman (2003.Arterioscler Thromb Vasc biol.23: 953-. In the present invention, the term "CD 31" refers more specifically to CD31 from a mammalian species, even more specifically to human CD 31. Preferably, the term "human CD 31" refers to the protein of amino acid sequence SEQ ID NO. 1, referenced by NP-000433 NCBI accession number. The amino acid numbering of human CD31 described herein corresponds to the amino acid numbering of the human CD31 sequence as set forth in SEQ ID NO:1 and is referenced by the NP-000433 NCBI accession number.

As used herein, "CD 38" refers to a 45kDa type II transmembrane glycoprotein also known as T10, cyclic ADP-ribose hydrolase 1(cyclic ADP-ribose hydrolases 1), ADPRC1, as described in Malavasi et al (2008.Physiological review.88: 841-886). In the present invention, the term "CD 38" refers more specifically to a polypeptide specifically from one mammalian species, even more specifically to human CD 38. Preferably, the term "human CD 38" refers to the protein of amino acid sequence SEQ ID NO. 2, referenced by NP-001766 NCBI accession number. The amino acid numbering of human CD38 as described herein corresponds to the amino acid numbering of the human CD38 sequence as set forth in SEQ ID NO:2 and is referenced by the NP-001766 NCBI accession number.

As used herein, "CDR" or "complementarity determining region" means a non-continuous antigen binding site found within the variable region of both heavy and light chain polypeptides. The identification of CDRs is according to the rules of Table 1 (deduced from Kabat et al, 1991.Sequences of proteins of immunological interest (5 th edition). Bethesda, MD: U.S. Dep.of Health and Human Services; and Chothia and Lesk,1987.J Mol biol.196(4):901-17), or by using the IMGT "collaier de rule" algorithm. In this respect, for the definition of the sequences of the invention, it is noted that the definition of the regions/domains may vary between different reference systems. Thus, the regions/domains described herein encompass sequences that vary by about +/-10% in length or location of the relevant sequence within the full-length sequence of the antibody variable domain.

TABLE 1

*Saul&Poljak,1992.Proteins.14(3):363-71

As used herein, "chimeric antibody" refers to an antibody in which a sequence of a variable domain derived from the germline of a mammalian species, such as a mouse, has been grafted onto a sequence of a constant domain derived from the germline of another mammalian species, such as a human. Advantageously, if the monoclonal antibody according to the invention is a chimeric monoclonal antibody, the latter comprises human constant regions. Chimeric antibodies can be prepared starting from non-human antibodies by using genetic recombination techniques well known to those skilled in the art. For example, chimeric antibodies can be produced by cloning heavy and light chain recombinant DNA comprising a promoter and sequences encoding the variable region of a non-human antibody, as well as sequences encoding the constant region of a non-human antibody. As a method for producing a chimeric antibody, for example, Verhoeyn et al (1988.science.239(4847):1534-6) can be referred to.

As used herein, "complement-dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system to an antibody that binds its cognate antigen. To assess complement activation, CDC assays are performed, for example, as described in Gazzano-Santoro et al, 1997, J Immunol methods.202(2): 163-71.

As used herein, "compound that specifically competes with CD 31" refers to any molecule suitable for pharmaceutical use that specifically binds to CD38, thereby shunting the interaction of endogenous CD31 with CD38 (bathing). Such compounds encompass antibodies, antigen-binding fragments thereof, antigen-binding antibody mimics, small organic molecules, oligonucleotides, and recombinant proteins.

As used herein with respect to a composition, pharmaceutical composition, or medicament, "consisting essentially of … …" means that the compound of the present invention is the only biologically active agent in the composition, pharmaceutical composition, or medicament.

As used herein, "epitope" refers to a particular arrangement of amino acids on one or more proteins to which an antibody or binding fragment thereof binds. Epitopes usually consist of chemically active surface groups of molecules, such as amino acids or sugar side chains, and have specific three-dimensional structural characteristics as well as specific charge characteristics. Epitopes may be linear (or contiguous) or conformational, i.e. involving two or more amino acid sequences in different regions of the antigen, which sequences may not necessarily be contiguous.

As used herein, a "fragment crystallizable region" or "Fc region" encompasses a polypeptide that comprises an antibody constant region and does not include a first constant region immunoglobulin domain. Thus, Fc refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, as well as the N-terminal flexible hinges for these domains.

For IgA and IgM, Fc may comprise J chains. For IgG, Fc comprises the immunoglobulin domains C γ 2 and C γ 3 and the hinge between C γ 1 and C γ 2.

Although the boundaries of the Fc region may vary, the Human IgG heavy chain Fc region is generally defined as comprising residues C226 or P230 at its carboxy terminus, with numbering according to the EU index as described in Kabat et al, 1991(Sequences of proteins of immunological interest (5 th edition). The EU index as described in Kabat refers to the residue numbering of the human IgG1EU antibody as described by Kabat et al, 1991 (supra). Fc may refer to this region in isolation, or in an antibody, antibody fragment, or Fc fusion protein.

The Fc fragment naturally consists of a C domain not included_H1, i.e. the lower boundary region and the constant domain C_H2 and C_H3 or C_H2 to C_H4 (depending on the isotype) constant region composition.

In the sense of the present invention, the Fc fragment of an antibody may be native or may be modified in various ways as long as it comprises a functional domain that binds to the FcR receptor (Fc γ R receptor for IgG) and preferably a functional domain that binds to the receptor FcRn. The modification may comprise the deletion of certain parts of the Fc fragment, provided that the latter comprises a domain that binds to the receptor FcR (for IgG the receptor fcyr), and preferably a domain that binds to the receptor FcRn. The modification of an antibody or of an antibody Fc fragment may also comprise various substitutions of amino acids capable of affecting the biological properties of the antibody, provided that the latter comprises a domain that binds to the receptor FcR, and preferably a domain that binds to the receptor FcRn.

In particular, when the antibody is an IgG, it may comprise a mutation aimed at enhancing binding to the receptor Fc γ RIII (CD16), as described in WO2000042072, WO2004029207, WO2004063351 or WO 2004074455. There may also be mutations that enhance binding to the receptor FcRn and thus enhance the in vivo half-life of the antibody, as described in e.g. WO2000042072, WO2002060919, WO2010045193 or WO 2010106180. Other mutations may or may not be present, such as those that reduce or increase binding to complement proteins and thus reduce or increase Complement Dependent Cytotoxicity (CDC) responses, for example as described in, for example, WO1999051642 or WO 2004074455.

As used herein, "Fc fusion protein" encompasses a polypeptide comprising an immunoglobulin Fc domain linked directly to another peptide or linked to another peptide using a linker.

As used herein, "framework region" or "FR" includes amino acid residues that are part of the variable region but are not part of the CDRs (e.g., using the Kabat/Chothia definition of CDRs). Thus, the variable region framework is between 100 and 120 amino acids in length, but includes only those amino acids outside the CDRs.

With respect to specific examples of HCVRs and the Kabat/Chothia-defined CDRs:

-FR1 may correspond to a domain encompassing amino acids 1-25 as defined according to Chothia/AbM, or encompassing the variable region of the next 5 residues as defined according to Kabat;

-FR2 may correspond to a domain encompassing the variable region of amino acids 36-49;

-FR3 may correspond to a domain encompassing the variable region of amino acids 67-98; and

FR4 may correspond to the domain of the variable region ending from amino acids 104-110 to the variable region.

The framework regions of the light chain are similarly separated by the CDRs of each LCVR. In naturally occurring antibodies, the six CDRs present on each monomeric antibody are short, discontinuous amino acid sequences that are specifically positioned to form an antigen binding site when the antibody assumes its three-dimensional configuration in an aqueous environment. The remainder of the heavy and light chain variable domains exhibit less intermolecular variability in amino acid sequence, termed framework regions. The framework regions adopt predominantly a β -sheet conformation, and the CDRs form loops that connect, and in some cases form part of, the β -sheet structure. Thus, these framework regions serve to form a scaffold that positions the six CDRs in the correct orientation by non-covalent interactions between the chains. The antigen binding site formed by the positioned CDRs determines the surface complementary to the epitope on the immunoreactive antigen. This complementary surface facilitates non-covalent binding of the antibody to the immunoreactive epitope. One of ordinary skill in the art can readily identify the location of the CDR.

As used herein, "heavy chain region" includes amino acid sequences derived from an immunoglobulin heavy chain constant domain. The protein comprising the heavy chain region comprises C_H1 domain, hinge (e.g., upper, middle and/or lower hinge region) domain, C_H2 domain, C_H3 domain or a variant or fragment thereof. In one embodiment, an antibody or binding fragment thereof of the invention can comprise an Fc region (e.g., hinge portion, C) of an immunoglobulin heavy chain_H2 domain and C_H3 domain). In another embodiment, an antibody or binding fragment thereof of the invention lacks at least a region of a constant domain (e.g., C)_H2 domain or all or part of a domain). In certain embodiments, at least one and preferably all of the constant domains are derived from a human immunoglobulin heavy chain. For example, in a preferred embodiment, the heavy chain region comprises a fully human hinge domain. In other preferred embodiments, the heavy chain region comprises a fully human Fc region (e.g., hinge, C from a human immunoglobulin)_H2 and C_H3 domain sequence). In certain embodiments, the constitutive constant domains of the heavy chain region are from different immunoglobulin molecules. For example, the heavy chain region of the protein may comprise a C derived from an IgGl molecule_H2 domain and a hinge region derived from an IgG3 or IgG4 molecule. In other embodiments, the constant domains are chimeric domains comprising regions of different immunoglobulin molecules. For example, the hinge may comprise a first region from an IgG1 molecule and a second region from an IgG3 or IgG4 molecule. As described above, one of ordinary skill in the art will appreciate that the constant domains of the heavy chain regions can be modified to differ in amino acid sequence from a naturally occurring (wild-type) immunoglobulin molecule. That is, the antibodies or binding fragments thereof of the invention may comprise a heavy chain constant domain(s) (C)_H1. Hinge, C_H2 or C_H3) And/or a light chain constant domain (C)_L) Change or modification of (a). Exemplary modifications include additions, deletions or substitutions of one or more amino acids in one or more domains.

As used herein, "hinge region" includesC is to be_H1 Domain is linked to C_H2 domain. This hinge region comprises about 25 residues and is flexible, thus allowing the two N-terminal antigen-binding regions to move independently. The hinge region can be subdivided into three distinct domains: upper, middle and lower hinge domains (Roux et al 1998.J Immunol.161(8): 4083-90).

As used herein, "human serum albumin fusion protein" refers to a recombinant protein consisting of a peptide fused to at least one domain of human serum albumin with or without a linker.

As used herein, "humanized antibody" refers to a chimeric antibody or binding fragment thereof that contains minimal sequence derived from a non-human immunoglobulin. It includes antibodies made from non-human cells having variable and constant regions that have been altered to more closely resemble antibodies made from human cells, for example, by altering the amino acid sequence of a non-human antibody to introduce amino acids found in human germline immunoglobulin sequences. The humanized antibodies or binding fragments thereof of the invention may comprise amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs. The term "humanized antibody" also includes antibodies and binding fragments thereof in which CDR sequences derived from the germline of another mammalian species (e.g., a mouse) have been grafted onto human framework sequences. In other words, the term "humanized antibody" refers to an antibody or binding fragment thereof in which the CDRs of an acceptor human antibody are replaced with CDRs from a donor non-human antibody (e.g., a mouse antibody). The humanized antibody or binding fragment thereof may also comprise donor-derived residues in the framework sequence. The humanized antibody or binding fragment thereof may further comprise at least a portion of a human immunoglobulin constant region. Humanized antibodies and binding fragments thereof may also comprise residues that are not found in either the recipient antibody or the imported CDR or FR sequences. Humanization can be performed using methods known in the art (e.g., Jones et al, 1986.Nature.321(6069): 522-5; Riechmann et al, 1988.Nature.332(6162): 323-7; Verhoeyen et al, 1988.science.239(4847): 1534-6; Presta,1992.Curr Opin Biotechnol.3(4): 394-8; patent U.S. Pat. No. 4,816,567), including techniques such as "Superhumanizing" antibodies (e.g., Tan et al, 2002.J Immunol.169(2):1119-25) and "resurfacing" (e.g., Staelens et al, 2006.Mol munol.43 (1994 8): 1243-57; Roguska et al, Proc Natl Acad. Sci.91 (3): 969-73).

Methods for humanizing the antibodies or binding fragments thereof of the present invention are well known in the art. The selection of light and heavy chain human variable domains for making humanized antibodies or binding fragments thereof is critical to reducing antigenicity. The sequences of the variable domains of the antibodies or binding fragments thereof according to the invention are screened against an entire library of known human variable domain sequences according to the so-called "best-fit" method. The human sequence closest to the mouse sequence was then taken up as the human Framework (FR) of the humanized antibody (Sims et al, 1993J Immunol.151(4): 2296-308; Chothia & Lesk, 1987J Mol biol.196(4): 901-17).

Another method of humanizing an antibody or binding fragment thereof according to the present invention uses specific FRs from the consensus sequence of all human antibodies from a particular subgroup of light or heavy chains. The same framework can be used for a variety of different humanized antibodies (Carter et al, 1992.Proc Natl Acad Sci USA.89(10): 4285-9; Presta et al, 1993.J Immunol.151(5): 2623-32). More importantly, the antibodies were humanized and retained high affinity for CD38 and other favorable biological properties. To achieve this goal, according to a preferred method, humanized antibodies and binding fragments thereof are prepared by an analytical process of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and familiar to those skilled in the art. A computer program can be used to illustrate and display the possible three-dimensional structures of selected candidate immunoglobulin sequences. Examination of these displays allows analysis of the likely role of the residues in the functional role (functionalization) of the candidate immunoglobulin sequence, i.e., analysis of residues that affect the ability of the candidate immunoglobulin to bind its epitope. In this way, CDR residues can be selected and combined from the consensus and import sequences to achieve a desired antibody characteristic, such as increased affinity for CD 38. Generally, CDR residues are directly and most substantially involved in affecting antigen binding.

Another method of humanizing the antibodies or binding fragments thereof of the present invention is to immunize a transgenic or transchromosomal animal carrying a portion of the human immune system. As hosts, immunoglobulin genes from these animals have been replaced by functional human immunoglobulin genes. Thus, antibodies produced by these animals or in hybridomas prepared from B cells from these animals have been humanized. Examples of such transgenic or transchromosomal animals include, but are not limited to:

-XenoMouse (Abgenix, Fremont, CA), described in patents US5,939,598, US6,075,181, US6,114,598, US6,150,584 and US6,162,963;

-HuMAb(Medarex, Inc.), described in Lonberg et al, 1994, Nature.368(6474): 856-859; lonberg&Huszar,1995.Int Rev Immunol.13(1):65-93；Harding&Lonberg,1995.Ann N Y Acad Sci.764: 536-46; taylor et al, 1992, Nucleic Acids Res.20(23): 6287-95; chen et al, 1993.Int Immunol.5(6): 647-56; tuaillon et al, 1993.Proc Natl Acad Sci USA.90(8): 3720-4; choi et al, 1993.Nat Genet.4(2): 117-23; chen et al, 1993, EMBO J.12(3): 821-30; tuaillon et al, 1994.J Immunol.152(6): 2912-20; taylor et al, 1994.Int Immunol.6(4): 579-91; fishwild et al, 1996.Nat Biotechnol.14(7): 845-51;

KM described in patent application WO2002043478

TC mice, described in Tomizuka et al, 2000.Proc Natl Acad Sci USA.97(2): 722-7;

-OmniRat^TM(OMT, Inc.) described in patent applications WO 2008151081; geurts et al, 2009.science 325(5939): 433; menoret et al, 2010 Eur J Immunol.40(10): 2932-41; osborn et al, 2013.J Immunol.190(4): 1481-90.

Humanized antibodies and binding fragments thereof can also be generated according to a variety of other techniques, such as by immunization using other transgenic animals (Jakobovitz et al, 1993.Nature.362(6417):255-8) that have been engineered to express a human antibody repertoire, or by selecting antibody repertoires using phage display methods. Such techniques are known to the skilled person and can be performed starting from the monoclonal antibodies or binding fragments thereof disclosed in the present application.

In some embodiments, an antibody or binding fragment thereof of the invention comprising a HCVR and a LCVR (or CDRs thereof) can comprise a first constant domain (C)_H1 and/or C_L) The amino acid sequence of which is wholly or substantially human.

In some embodiments, particularly when the antibodies or binding fragments thereof of the invention are intended for therapeutic use in humans, the entire constant region, or at least a portion thereof, typically has a fully or substantially human amino acid sequence. Thus, C_H1 domain, hinge region, C_H2 domain, C_H3 domains and C_LDomains (and C)_H4 domain, if present) or any combination thereof may be fully or substantially human with respect to their amino acid sequence. Advantageously, C_H1 domain, hinge region, C_H2 domain, C_H3 domains and C_LDomains (and C)_H4 domains, if present) can each have a fully or substantially human amino acid sequence.

The term "substantially human" in the context of a constant region of a humanized or chimeric antibody or binding fragment thereof refers to a constant region that has at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity to a human constant region.

As used herein, the term "human amino acid sequence" refers to an amino acid sequence encoded by a human immunoglobulin gene, which includes germline, rearranged and somatically mutated genes. The invention also relates to proteins comprising constant domains of "human" sequences, which have been altered by one or more amino acid additions, deletions or substitutions relative to the human sequence, except for those embodiments in which the presence of a "fully human hinge region" is explicitly required.

The presence of a "fully human hinge region" in the antibodies or binding fragments thereof of the invention may be beneficial for both minimizing immunogenicity and optimizing antibody stability. It is believed that one or more amino acid substitutions, insertions or deletions may be made in the constant region of the heavy and/or light chain, particularly in the Fc region. Amino acid substitutions can result in the substitution of a substituted amino acid with a different naturally occurring amino acid, or an unnatural or modified amino acid. Other structural modifications are also permissible, such as changes in the glycosylation pattern (e.g., by addition or deletion of N-or O-linked glycosylation sites). Depending on the intended use of the antibody or binding fragment thereof, it may be desirable to modify the antibody or binding fragment thereof of the invention with respect to its binding properties to an Fc receptor, e.g., modulating effector function. For example, cysteine residues may be introduced into the Fc region, allowing interchain disulfide bonds to form in this region. The homodimeric antibodies thus produced may have improved effector function (Caron et al, 1992.J Exp Med.176(4): 1191-5; shop, 1992.J Immunol.148(9): 2918-22).

As used herein, "hypervariable loops" are not strictly synonymous with Complementarity Determining Regions (CDRs) due to the fact that hypervariable loops (HV) are defined based on structure, whereas CDRs are defined based on sequence variability (Kabat et al, 1991.Sequences of proteins of immunological interest (5 th edition). Bethesda, MD: U.S. Dep.of Health and Human Services), and the limitations of HV and CDRs are in some V_HAnd V_LThe domains may differ. V_LAnd V_HThe CDRs of a domain can generally be defined by the Kabat/Chothia definition already explained above.

"identity" or "identical" when used in relation between two or more amino acid sequences, or sequences of two or more nucleic acid sequences, refers to the degree of sequence relatedness between amino acid sequences or nucleic acid sequences, as determined by the number of matches between two or more amino acid residues or fragments of nucleic acid residues (string). "identity" measures the percentage of identical matches between smaller sequences of two or more sequences having gap alignments (if any) that are processed by a particular mathematical model or computer program (i.e., an "algorithm").

The identity of related amino acid sequences or nucleic acid sequences can be readily calculated by known methods. Such methods include, but are not limited to, those described below: lesk A.M, (1988) computerized molecular biology: Sources and methods for sequence analysis. New York, NY: Oxford University Press; smith D.W, (1993) Biocomputing: information and genome projects.san Diego, CA: Academic Press; griffin A.M. & Griffin H.G. (1994). Computer analysis of sequence data, Part 1.Totowa, NJ: Humana Press; von Heijne G. (1987). Sequence analysis in molecular biology: treasuretrove or triviral drive.san Diego, CA: Academic press; gribskov M.R. & Devereux J. (1991.) Sequence analysis primer.New York, NY: Stockton Press; carillo et al, 1988, SIAM J Appl Math.48(5): 1073-82.

The preferred method of determining identity is designed to provide the largest match between test sequences. Methods of determining identity are described in publicly available computer programs. Preferred Computer program methods for determining identity between two sequences include the GCG package, including GAP (Genetics Computer Group, University of Wisconsin, Madison, Wis.; Devereux et al, 1984.Nucleic Acids Res.12(1Pt 1):387-95), BLASTP, BLASTN, and FASTA (Altschul et al, 1990.J Mol biol.215(3): 403-10). The BLASTX program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST handbook, Altschul et al NCB/NLM/NIH Bethesda, Md.20894). The well-known Smith Waterman algorithm (Smith Waterman algorithm) may also be used to determine identity.

As used herein, "pharmaceutical" is intended to encompass compositions suitable for administration to a subject or patient, e.g., a mammal, especially a human. Typically, a "drug" is sterile and typically free of contaminants that can cause an undesirable response in a subject (e.g., the compounds in the drug are pharmaceutical grade). The drug may be designed to be administered to a subject in need thereof by a variety of different administration routes including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, subcutaneous, intranasal, intrathecal, perimedullary (periprepinal), and the like. The preferred form depends on the intended mode of administration and therapeutic application. Typical preferred forms are oral solutions, injectable solutions or infusible solutions.

As used herein, a "modified antibody" corresponds to a molecule comprising an antibody or antigen-binding fragment thereof, wherein the antibody or fragment thereof is bound to a functionally distinct molecule. The modified antibodies of the invention may be fusion chimeric proteins or conjugates produced by any suitable form of linkage, including covalent attachment (grafting), chemical bonding with a chemical or biological group or molecule such as a PEG polymer or another protective group or molecule suitable for preventing protease cleavage in vivo, to improve the stability and/or half-life of the antibody or functional fragment. Using similar techniques, especially by chemical ligation or grafting, modified antibodies can be prepared with biologically active molecules selected, for example, from the a chain of toxins, especially pseudomonas exotoxin a (pseudomonas exotoxin a), the phytotoxin ricin (ricin) or saporin (saporin) toxins, especially therapeutically active ingredients, carriers (including especially protein carriers) suitable for targeting the antibodies or functional fragments to specific cells or tissues of the human body, or they can be conjugated with labels or with linkers, especially when fragments of antibodies are used. PEGylation (PEGylation) of an antibody or a functional fragment thereof is a particularly interesting embodiment, since it improves the delivery conditions of the active substance to the host, especially for therapeutic applications. Pegylation may be site-specific to prevent interference with the recognition site of the antibody or functional fragment, and may be performed using high molecular weight PEG. Pegylation can be achieved by free cysteine residues present in the sequence of the antibody or functional fragment or by free cysteine residues added to the amino acid sequence of the antibody or functional fragment.

As used herein, "monoclonal antibody" is intended to refer to a preparation of antibody molecules having common heavy and common light chain amino acid sequences, as opposed to a preparation of "polyclonal antibodies" that contain a mixture of antibodies of different amino acid sequences. Certain differences in protein sequence associated with post-translational modifications (e.g., cleavage of heavy chain C-terminal lysine, deamidation of asparagine residues, and/or isomerization of aspartic acid residues) may still exist between the individual antibodies present in the monoclonal antibody composition. The modifier "monoclonal" is not to be construed as requiring production of the antibody by any particular method. Monoclonal antibodies can be produced by a variety of known techniques, such as phage, bacterial, yeast or ribosome display, as well as the classical approach exemplified by hybridoma-derived antibodies, which was first described by Kohler et al (1975.Nature.256(5517): 495-7). Thus, the term "monoclonal" is used to refer to all antibodies derived from only a single nucleic acid clone.

As used herein, "oligonucleotide" refers to short molecules of nucleotides, including DNA, RNA, and modified nucleotides (e.g., nucleotides comprising at least one chemical modification). In particular, the oligonucleotide consists of less than 200 nucleotides, more particularly less than 50 nucleotides.

As used herein, "oligonucleotide aptamer" refers to a short oligonucleotide that is capable of selectively binding a small molecule ligand or protein target with high affinity and specificity when folded into its unique three-dimensional structure.

As used herein, "peptide" is intended to refer to a molecule consisting of at least amino acids. The peptides may also have other molecular groups, such as polysaccharide chains or other post-translational modifications. "recombinant peptide" specifically refers to a peptide that is produced, expressed, produced, or isolated by recombinant means, e.g., expressed using a recombinant expression vector transfected into a host cell. Recombinant peptides include, for example, chimeric peptides. "chimeric peptide" is intended to mean a peptide obtained by linking two or more genes originally encoding different proteins or protein fragments; or a peptide produced by fusing two or more proteins or protein fragments. Chimeric peptides include, for example, peptides fused to the Fc region of IgG, to a specific domain of human serum albumin (HAS) or HSA (e.g., domain III), or transferrin (e.g., Strohl,2015.BioDrugs.29(4): 215-239).

As used herein, "peptide aptamer" refers to a small synthetic protein that is selected to bind to a specific site on its target antigen.

As used herein, "recombinant antibody" refers to an antibody produced, expressed, produced, or isolated by recombinant means, e.g., an antibody expressed using a recombinant expression vector transfected into a host cell; antibodies isolated from a recombinant combinatorial antibody library; antibodies isolated from transgenic animals (e.g., mice) for human immunoglobulin genes; or antibodies produced, expressed, generated or isolated in any other manner in which a particular immunoglobulin gene sequence (e.g., a human immunoglobulin gene sequence) is assembled with other DNA sequences. Recombinant antibodies include, for example, chimeric antibodies and humanized antibodies.

As used herein, "small organic molecule" refers to a low molecular weight organic compound (up to 5000Da, more specifically up to 2000Da, and most specifically up to about 1000 Da).

As used herein, "subject" refers to a mammal, preferably a human. In one embodiment, the subject may be a "patient", i.e. a warm-blooded animal, more preferably a human, who is awaiting the receipt or is receiving medical care or was/is/will be the subject of a medical procedure, or is being monitored for the development of a disease. The term "mammal" as used herein refers to any mammal, including humans, domestic and farm animals, as well as zoo, sports or pet animals, such as dogs, cats, cows, horses, sheep, pigs, goats, rabbits, and the like. Preferably, the mammal is a primate, more preferably a human.

As used herein, "therapeutically effective amount" means the level, amount, or concentration of an agent (e.g., a compound that competes with CD31 for binding to CD 38) that targets: (1) delaying or preventing the onset of a target disease; (2) slowing or arresting the development, exacerbation or worsening of one or more symptoms of the target disease; (3) ameliorating a symptom of the target disease; (4) reducing the severity or incidence of a target disease; (5) curing the target disease. A therapeutically effective amount may be administered prior to the onset of the targeted disease for prophylactic or preventative effects. Alternatively or additionally, a therapeutically effective amount may be administered after the onset of the targeted disease for therapeutic effect.

As used herein, "treatment" or "therapy" or "alleviation" refers to both therapeutic and prophylactic (or preventative) measures; wherein the goal is to slow (or alleviate) the target disease. Subjects in need of treatment include subjects already having the disease of interest and subjects suspected of having the disease of interest.

As used herein, "variable region" or "variable domain" refers to the variable domain V_HAnd V_LThe sequence of certain regions of (a) differ greatly between antibodies and is used for the binding and specificity of each particular antibody for its target antigen. However, the variability is not evenly distributed throughout the antibody variable domain. It concentrates on every V_LDomains and V_HThree segments of the domain, termed "hypervariable loops", form part of the antigen-binding site. The first, second, and third hypervariable loops of the V λ light chain domain are referred to herein as L1(λ), L2(λ), and L3(λ), and may be defined as comprising V_LResidues 24-33(L1 (. lamda.) consisting of 9, 10 or 11 amino acid residues), residues 49-53(L2 (. lamda.) consisting of 3 residues) and residues 90-96(L3 (. lamda.) consisting of 6 residues) in the domain (Morea et al, 2000.methods.20(3): 267-79).

The first, second and third hypervariable loops of the vk light chain domain are referred to herein as L1(κ), L2(κ) and L3(κ) and may be defined as comprising V_LResidues 25-33(L1(κ), consisting of 6, 7, 8, 11, 12 or 13 residues), residues 49-53(L2(κ), consisting of 3 residues) and residues 90-97(L3(κ), consisting of 6 residues) in the domain (Morea et al, 2000.methods.20(3): 267-79).

V_HThe first, second and third hypervariable loops of the domain are referred to herein as H1, H2 and H3 and may be defined as comprising V_HResidues 25-33(H1, consisting of 7, 8 or 9 residues), residues 52-56(H2, consisting of 3 or 4 residues) and residues 91-105(H3, highly variable in length) in the domain (Morea et al, 2000.methods.20(3): 267-79).

Unless otherwise indicated, the terms L1, L2, and L3 refer to V, respectively_LFirst, second and third hypervariable loops of the domain,and encompasses hypervariable loops obtained from the vk and V λ isoforms. The terms H1, H2 and H3 refer to V, respectively_HFirst, second and third hypervariable loops of the domain and encompasses hypervariable loops obtained from any known heavy chain isotype, including gamma (γ), mu (μ), alpha (α), delta (δ) or epsilon (ε). Hypervariable loops L1, L2, L3, H1, H2 and H3 may each comprise a portion of a "complementarity determining region" or "CDR" as described above.

Brief description of the drawings

The diagram of fig. 1 shows sCD31 for MPP⁺Neuroprotective effects of induced Dopaminergic (DA) cell death. MPP between 5 and 7 DIVs with or without sCD31(0.01, 0.03, 0.1, 0.3, 1. mu.g/ml) exposure⁺DA (TH) in (3. mu.M) treated midbrain cultures⁺) Cell viability. Results are expressed as a percentage of the corresponding control culture. $ P<0.05 one-way ANOVA compared to control treatment; # P<0.05 and MPP⁺Treatment of comparative one-way ANOVA.

FIG. 2 is a bar graph showing sCD31 for MPP⁺Neuroprotective effects of induced DA cell death. MPP was used between 5 to 7 DIVs exposed or not exposed to sCD31 (1. mu.g/ml) in the presence or absence of anti-CD 31 antibody clone Moon-1 (1. mu.g/ml), antagonistic anti-CD 38 antibody clone OKT10(OKT, 1. mu.g/ml) or antagonistic anti-CD 38 antibody clone AT13/5 (1. mu.g/ml)⁺DA (TH) in (3. mu.M) treated midbrain cultures⁺) Cell viability. Results are expressed as a percentage of the corresponding control culture. $ P<0.05 one-way ANOVA compared to control treatment; # P<0.05 and MPP⁺Treatment of comparative one-way ANOVA.

FIG. 3 is a bar graph showing agonistic anti-CD 38 antibody clone HB7 against MPP⁺Neuroprotective effects of induced DA cell death. MPP was used between 5 and 7 DIVs exposed or not exposed to an agonistic anti-CD 38 antibody (clone HB7, 1. mu.g/ml) in the presence or absence of antagonistic antibody clone OKT10(OKT, 1. mu.g/ml) or antagonistic anti-CD 38 antibody clone AT13/5 (1. mu.g/ml)⁺DA (TH) in (3. mu.M) treated midbrain cultures⁺) Cell viability. Results are expressed as a percentage of the corresponding control culture. $ P<0.05 one-way ANOVA compared to control treatment; # P<0.05 and MPP⁺Treatment of comparative one-way ANOVA.

FIG. 4 is a bar graph showing that sCD31 or agonistic anti-CD 38 antibody clone HB7 is directed against MPP in the presence or absence of the tyrosine kinase inhibitor genistein⁺Neuroprotective effects of induced DA cell death. MPP was used between 5 and 7 DIVs with or without sCD31 (1. mu.g/ml) or agonistic anti-CD 38 antibody (clone HB7, 1. mu.g/ml) in the presence or absence of the tyrosine kinase inhibitor genistein (GENI, 10nM)⁺DA (TH) in (3. mu.M) treated midbrain cultures⁺) Cell viability. Results are expressed as a percentage of the corresponding control culture. $ P<0.05 one-way ANOVA compared to control treatment; # P<0.05 and MPP⁺Treatment of comparative one-way ANOVA.

FIG. 5 is a bar graph showing that sCD31 or agonistic anti-CD 38 antibody clone HB7 is directed against MPP in the presence or absence of lysosomal exocytosis inhibitors vacuolin-1 or endosidin2⁺Neuroprotective effects of induced DA cell death. MPP was used between 5 and 7 DIVs with or without sCD31(1 μ g/ml) or agonistic anti-CD 38 antibody (clone HB7, 1 μ g/ml) in the presence or absence of two lysosomal maturation and exocytosis inhibitors, vacuolin-1(VAC, 10 μ M) or endosidin2(40 μ M)⁺DA (TH) in (3. mu.M) treated midbrain cultures⁺) Cell viability. Results are expressed as a percentage of the corresponding control culture. $ P<0.05 one-way ANOVA compared to control treatment; # P<0.05 and MPP⁺Treatment of comparative one-way ANOVA.

FIG. 6 is a bar graph showing the effect of an intravenously injected agonistic anti-CD 38HB7 antibody (15mg/kg iv, two days before MPTP injection) on DA (TH) in the substantia nigra pars compacta (substanta nigra pars compact in an in vivo MPTP mouse model⁺) Influence of the number of neurons. Results are expressed as a percentage of control (PBS injected) mice. PBS group: n is 5; MPTP group: n is 6; MPTP + HB 715 mg/kg group iv: n is 8. $ P<0.05 one-way ANOVA compared to control treatment. # P<0.05 and MPP⁺Treatment of comparative one-way ANOVA.

FIG. 7 is a bar graph showing the effect of intravenous injection of the agonistic anti-CD 38 clone HB7 on IL-10 levels in mice. P <0.05 one-way ANOVA compared to control (PBS injected) group.

Figure 8 is a graph depicting a process for identifying anti-CD 38 antibodies that compete with CD 31.

FIG. 9 is a bar graph showing novel humanized anti-CD 38 antibodies (clones A02, B06, B08, B09, C05, C06, C08, D03, D04, D06, D07, D10, E03, E05, E08, F07) (identified by phage display) or anti-CD 38 antibody clone HB7 against MPP⁺Neuroprotective effects of induced DA cell death. MPP was used between 5 to 7 DIVs with or without exposure to anti-CD 38 antibody (clones A02, B06, B08, B09, C05, C06, C08, D03, D04, D06, D07, D10, E03, E05, E08, F07; 0.5. mu.g/ml) or anti-CD 38 antibody clone HB7 (0.5. mu.g/ml)⁺DA (TH) in (3. mu.M) treated midbrain cultures⁺) Cell viability. Results are expressed as a percentage of the corresponding control culture. # P<0.05 and MPP⁺Treatment of comparative one-way ANOVA.

FIG. 10 is a bar graph showing novel humanized anti-CD 38 antibodies (clones B08, C05, C06, D06 and D07) identified by phage display against MPP in the presence or absence of the lysosomal exocytosis inhibitor vacuolin-1⁺Neuroprotective effects of induced DA cell death. MPP was used between 5 and 7 DIVs with or without exposure to anti-CD 38 antibodies (clones B08, C05, C06, C08, D06 and D07; 1. mu.g/ml) in the presence or absence of the lysosomal exocytosis inhibitor vacuolin-1(VAC, 10. mu.M)⁺DA (TH) in (3. mu.M) treated midbrain cultures⁺) Cell viability. Results are expressed as a percentage of the corresponding control culture. $ P<0.05 one-way ANOVA compared to control treatment; # P<0.05 and MPP⁺Treatment of comparative one-way ANOVA.

FIG. 11 is a bar graph showing the intracerebral injection (0.1mg/kg icv, simultaneously with 6-OHDA icv injection) of anti-CD 38 antibody (clones HB7, B08 or D06) against DA (TH) in the substantia nigra pars compacta in an in vivo 6-OHDA mouse model⁺) Influence of the number of neurons. Results are expressed as a percentage of contralateral (non-diseased). PBS group: n-8 (PBS); 6-OHDA + PBS group: n-8 (PBS); 6-OHDA + HB70.1mg/kg icv group: n-10 (HB7icv 0.1 mg/kg); 6-OHDA + B080.1mg/kg icv group: n-7 (B8icv 0.1 mg/kg); 6-OHDA + D060.1mg/kg icv group:n＝7(D6icv 0.1mg/kg)。$P<0.05 one-way ANOVA compared to control (PBS injected) group. # P<0.05 one-way ANOVA compared to the 6-OHDA group.

FIG. 12 is a bar graph showing the intravenous injection (15mg/kg iv, day before 6-OHDAIcv) of anti-CD 38 antibody (clone B08 or Daratumumab (Daratumumab), DARA) on DA (TH) in the substantia nigra dense in vivo 6-OHDA mouse model⁺) Influence of the number of neurons. Results are expressed as a percentage of contralateral (non-diseased). PBS group: n-8 (PBS); 6-OHDA group: n-8 (PBS); 6-OHDA + B0815 mg/kg group iv: n-10 (B8iv 15 mg/kg); 6-OHDA + Daranda monoclonal antibody 15mg/kg group iv: n is 10(DARA iv 15 mg/kg). $ P<0.05 one-way ANOVA compared to control (PBS injected) group. # P<0.05 one-way ANOVA compared to the 6-OHDA group.

FIG. 13 is a bar graph showing the intravenous injection (15mg/kg iv, two days after 6-OHDA icv injection) of B08 anti-CD 38 antibody against DA (TH) in the substantia nigra pars compacta in an in vivo 6-OHDA mouse model⁺) Influence of the number of neurons. Results are expressed as a percentage of contralateral (non-diseased). PBS group: n-12 (PBS); 6-OHDA + PBS group: n-12 (PBS); 6-OHDA + B0815 mg/kg group iv: n-12 (B8iv 15 mg/kg). $ P<0.05 one-way ANOVA compared to control (PBS injected) group. # P<0.05 one-way ANOVA compared to the 6-OHDA group.

FIG. 14 is a bar graph showing the effect of using human peripheral blood mononuclear cells from 3 healthy donors, an anti-CD 38 antibody (clone HB7, B08, C05, D06 or D07) on anti-inflammatory cytokine IL-10 release (pooled results). $ P <0.05 one-way ANOVA compared to control (PBS-treated) group.

Fig. 15A-B are a set of schematic and graph diagrams. (A) The schematic representation of (A) represents the experimental protocol. (B) Bar graphs show the effect of intravenous injection (15mg/kg iv, 10 and 20 days post MOG injection) of anti-CD 38 antibody (clone B08) on clinical scores in an in vivo EAE mouse model. PBS group: n-10 (PBS); group B0815 mg/kg iv: n is 10 (B815 mg/kg iv). P <0.05 two-way ANOVA compared to PBS group. P <0.01 two-way ANOVA compared to PBS group. Clinical scores were calculated as described in example 9, section 2.4 b).

Fig. 16A-B are a set of schematic and graph diagrams. (A) The schematic representation of (a) represents the experimental protocol. (B) Bar graphs of (B) show the effect of intravenous injection (15mg/kg iv at D0) of anti-CD 38 antibody (clone B08 or daratumumab, DARA) on colon length in an in vivo DSS mouse model. No DSS group: n-5 (PBS; white bar); DSS + PBS group: n-6 (PBS; black bars); DSS + DARA 15mg/kg iv group: n-7 (DARA iv 15 mg/kg; black bars); DSS + B0815 mg/kg group iv: n-6 (B8iv 15 mg/kg; black bars). $ P <0.05 one-way ANOVA compared to no DSS group. # P <0.05 one-way ANOVA compared to DSS group.

Figure 17 is a bar graph showing plasma CD38 levels in human Healthy Control (HC) or Amyotrophic Lateral Sclerosis (ALS) patients. HC group: n-46; ALS group: n is 40. P <0.001, t-test compared to HC group.

Examples

The present invention is further illustrated in the following examples, but the technical scope of the present invention is not limited to these examples.

Example 1: the in vitro neuroprotective effect of sCD31 on mesencephalic dopaminergic neurons is mediated through interaction with CD38

⁺Protection against mitochondrial neurotoxin MPP

We report here that the death of DA cells is caused by the active metabolite 1-methyl-4-phenylpyridine (MPP) of the DA neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPP)⁺) Under the condition caused by mitochondrial poisoning, sCD31 has a neuroprotective effect.

For this purpose, by combining the depolarization concentrations of K⁺(30mM) and MK801(5 μ M), a glutamate receptor antagonist used to prevent unwanted excitotoxic damage, to prevent spontaneous DA cell death. Cultures were then exposed to 3 μ M MPP for 5 to 7 Days In Vitro (DIV)⁺To achieve approximately 50% loss of DA neurons. This compound (FIG. 1) had an EC of 36ng/mL when cultures were exposed to murine sCD31 (having SEQ ID NO: 3) during intoxication₅₀Prevent MPP⁺Induced DA cell loss.

Notably, these effects are true and direct neuroprotective effects and are not due to possible interference of CD38 on immune cells (as described in Hara-Yokoyama et al, 2008.Int Immunopharmacol.8(1): 59-70), since there are no peripheral immune cells in this in vitro cell culture environment.

Neuroprotective Effect of sCD31 in the anti-CD 31 antibody clone Moon-1 or antagonistic anti-CD 38 antibody (clone OKT- 10 or AT13/5) are antagonized

CD31 is known to interact with the following 3 ligands: CD31 itself, CD38 and α V β 3 integrin (Kalinowska & Losy,2006.Eur J neurol.13(12): 1284-90). However, CD31 is not expressed in neuronal cells (Williams et al, 1996.J Neurosci Res.45(6): 747-57). The interaction of CD31 with CD38 has previously been shown to be antagonized in the presence of the anti-CD 31 antibody clone Moon-1 or antagonistic anti-CD 38 antibody (Deaglio et al, 1996.J Immunol.156(2): 727-34; Deaglio et al, 1998.J Immunol.160(1): 395-402).

To test whether the neuroprotective effect of sCD31 was mediated by CD38, we previously described MPP⁺The neuroprotective effect of sCD31 (1. mu.g/mL) in the presence or absence of anti-CD 31 antibody clone Moon-1 (1. mu.g/mL) or antagonistic anti-CD 38 antibody (clone OKT10 or AT13/5, 1. mu.g/mL) was studied in an in vitro model. .

We observed that the neuroprotective effect of sCD31 was antagonized in the presence of anti-CD 31 antibody clone Moon-1 or antagonistic anti-CD 38 antibody clone OKT10 or AT13/5, thus implying that the neuroprotective effect of sCD31 is mediated by interaction with CD38 (fig. 2).

The neuroprotective effect of sCD31 was reproduced by an agonistic anti-CD 38 antibody (clone HB7)Agonistic anti-CD 38 antibodies are compounds that reproduce the biological effects of CD31 binding to CD38 (Deaglio et al, 1998.J Immunol.160(1): 395-402). Thus, like sCD31, agonistic anti-CD 38 antibodies should also have neuroprotective effects.

We have observed that at MPP⁺In vitro model, agonistic anti-CD 38 antibody (clone HB7, 1. mu.g/mL) was able to protect DA neurons, and this neuroprotection was performedAntagonized in the presence of an antagonistic anti-CD 38 antibody (clone OKT10 or AT13/5, 1. mu.g/mL) (FIG. 3).

Neuroprotective effects of sCD31 or agonist anti-CD 38 antibodies require tyrosine phosphorylation/agonism of tyrosine kinases Activity device

As described above, CD38 is a multifunctional molecule that includes receptor-mediated functions (via internalization), tyrosine phosphorylation-mediated functions, and enzyme-mediated functions. Previous studies have shown that agonistic (clone HB7) or antagonistic (clone OKT10 or AT13/5) anti-CD 38 antibodies are unable to modulate CD38 enzyme activity in vitro (Deckert et al, 2014.Clin Cancer Res.20(17): 4574-83). Therefore, we wanted to determine whether sCD31 or agonistic anti-CD 38 antibodies require tyrosine phosphorylation to mediate their neuroprotective effects by using the tyrosine kinase inhibitor genistein.

We have observed that at MPP⁺In an in vitro model, the neuroprotective effects of sCD31 (1. mu.g/mL) or agonistic anti-CD 38 antibody (clone HB7, 1. mu.g/mL) were antagonized in the presence of the tyrosine kinase inhibitor genistein (GENI, 10nM), indicating that tyrosine phosphorylation is necessary for the protection of DA neurons (FIG. 4).

Neuroprotective effects of sCD31 or agonistic anti-CD 38 antibodies are mediated by lysosomal exocytosis

It is likely that both sCD31 and agonistic anti-CD 38 antibodies may protect dopaminergic neurons by acting on CD38 levels but by different mechanisms of action. Excluding this possibility, we observed (FIG. 5) at MPP⁺Neuroprotective effects of sCD31 (1. mu.g/mL) or agonistic anti-CD 38 antibody (clone HB7, 1. mu.g/mL) in vitro models at lysosomal maturation inhibitors and Ca²⁺Both the lysosome dependent exocytosis inhibitors vacuolin-1 and endosidin2 were antagonized.

Agonistic anti-CD 38 antibody (clone HB7) has neuroprotective effect in vivo

To determine whether an agonistic anti-CD 38 antibody (clone HB7) also had neuroprotective effects in vivo, we injected this anti-CD 38 intravenously at a dose of 15mg/kgIn vivo in a mouse model, dopaminergic neurons of the substantia nigra pars compacta are denatured following repeated intraperitoneal injections of MPTP. MPTP has the ability to cross the blood brain barrier and is converted into MPP by astrocytes⁺。MPP⁺Are inhibitors of mitochondrial complex I which are preferentially transported in dopaminergic neurons by dopamine transporters, leading to specific cell death in dopaminergic populations. This model has been widely used as a test system to evaluate neuroprotection and neurorepair strategies (Dauer)&Przedborski,2003.Neuron.39(6):889-909)。

We observed that intravenous injection of an agonistic anti-CD 38 antibody (clone HB7) at a dose of 15mg/kg strongly protected dopaminergic neurons of the substantia nigra pars compacta from MPTP-induced neurodegeneration (fig. 6).

Conclusion

In summary, the data provided in this example show that sCD31 has a neuroprotective effect in vitro, mediated through interaction with CD 38. This effect is antagonized in the presence of an anti-CD 31 antibody or an antagonistic anti-CD 38 antibody.

Furthermore, agonistic anti-CD 38 antibodies that mimic binding of sCD31 to CD38 have also been shown to be able to induce neuroprotective effects that are antagonized in the presence of antagonistic anti-CD 38 antibodies.

Whether neuroprotection is induced by sCD31 or by agonistic anti-CD 38 antibodies, tyrosine phosphorylation/activation of tyrosine kinases has been shown to be essential for the induction of such neuroprotection. Furthermore, it has been shown that the neuroprotective effect produced is due to lysosomal maturation and Ca²⁺Dependent lysosomal exocytosis.

Finally, agonistic anti-CD 38 antibody was shown to have neuroprotective effects in vivo using the intravenous route of administration in a widely used MPTP mouse model, demonstrating that agonistic anti-CD 38 antibody is able to cross the BBB and engage its target (engage).

Thus, these data indicate therapeutic roles for sCD31 and agonistic anti-CD 38 antibodies in the treatment of neurodegenerative and neuroinflammatory diseases.

Example 2: in vivo Effect of sCD31 or agonistic anti-CD 38 antibody (clone HB7) on IL-10 levels

Agonistic anti-CD 38 antibody (clone HB7) increases IL-10 in vivo

To examine whether the anti-inflammatory properties of the agonistic anti-CD 38 antibody were also observed in vivo, we injected mice intravenously with anti-CD 38 antibody clone HB7(15 mg/kg).

Eight days after injection, we observed a 6-fold increase in IL-10 levels (FIG. 7).

Conclusion

In summary, the data presented in this example show that both sCD31 and agonistic anti-CD 38 antibodies induce strong release of IL-10 in vivo, suggesting a role in immune regulation and anti-inflammation. Indeed, it is well known that IL-10 causes T on macrophages_h1 down-regulation of the expression of cytokines, MHC class II antigens and co-stimulatory molecules; enhancing B cell survival, proliferation and antibody production; block NF-kB activity; and regulates the JAK-STAT signal pathway.

In addition, IL-10 has also been shown to be effective in treating cancer, particularly by inhibiting tumor metastasis (Sun et al, 2000.J Immunother.23(2): 208-14). IL-10 expression from transfected tumor cell lines in IL-10 transgenic mice (group et al, 1999.J Immunol.162(3):1723-9) or IL-10 administration resulted in controlled primary tumor growth and reduced metastatic burden (Fujii et al, 2001.blood.98(7): 2143-51; Berman et al, 1996.J Immunol.157(1): 231-8).

Thus, these data indicate therapeutic roles for sCD31 and agonistic anti-CD 38 antibodies in the treatment of inflammatory diseases and cancer.

Example 3: identification of agonistic anti-CD 38 antibodies by phage display that compete with CD31

To generate new humanized antibodies that reproduce the biological effect of CD31 on CD38, we used a scFv phage display library containing 15 hundred million sequences (Philibert et al, 2007.BMC biotechnol.7: 81).

A first round of selection was performed to identify scFv that bound the extracellular domain of human CD38 (figure 8). Of the 95 selected scfvs, 52 were confirmed by FACS to be associated with CD38⁺Jurkat T cell binding. These 52 validated binders (binders) were sequenced, thereby identifying 16 non-redundant scfvs (non-redundant scfvs) that bind CD 38. These binders were cloned and the corresponding IgG was produced in CHO cells.

16 non-redundant iggs (clones a02, B06, B08, B09, C05, C06, C08, D03, D04, D06, D07, D10, E03, E05, E08, F07) were then tested as follows: firstly, at the MPP⁺In an in vitro model (0.5. mu.g/mL per clone), competition assays for a panel of selected antibodies with the human extracellular domain of CD31 were then performed by Biacore.

At MPP⁺In vitro models, several humanized anti-CD 38 antibody clones were found to have neuroprotective effects (fig. 9). Specifically, 4 clones (B08, C05, D06 and D10) showed stronger neuroprotective effects than the reference agonistic anti-CD 38 antibody clone HB7(0.5 μ g/mL).

The neuroprotective effects of clones B08, C05, C06, D06 and D07 were antagonized in the presence of the lysosomal exocytosis inhibitor vacuolin-1(VAC, 10 μ M), suggesting that these antibodies reproduce the biological effects of sCD31 (fig. 10).

Example 4: neuroprotective properties of B08 and C06 anti-CD 38 antibodies in an in vivo neurodegenerative model

We aimed to determine whether the B08 or D06 anti-CD 38 antibodies have neuroprotective effects against oxidative stress-induced neurodegeneration in vivo. 6-hydroxydopamine (6-OHDA) was administered to the right striatum (striatum) of mice. Since 6-OHDA is a dopamine analog, it is transported in dopaminergic neurons by dopamine transporters, resulting in specific degeneration of the right substantia nigra pars compacta and loss of dopaminergic neurons. It has been widely used as a New symptomatic agent (symptomatic agent) and as a test system for the evaluation of neuroprotective and neurorestorative strategies (Galindo et al 2014.Kostrzewa, Handbook of neurooxidity (1 st edition, page 639-651.) New York: Springer-Verlag). Since 6-OHDA was injected unilaterally into the right striatum, the left striatum was unaffected and each mouse was its own control.

Mice were injected intracerebrally with 0.1mg/kg of HB7, B08, and D06 anti-CD 38 antibodies. We observed that B08 and D06 anti-CD 38 antibodies statistically significantly protected dopaminergic neurons of the right substantia nigra pars compacta when injected simultaneously with 6-OHDA intracerebrally (icv) at a concentration of 0.1mg/kg (fig. 11). We also observed that HB7 anti-CD 38 antibody failed to protect dopaminergic neurons statistically significantly.

Interestingly, with respect to the B08 anti-CD 38 antibody, this neuroprotective effect was also observed when injected intravenously one day before the 6-OHDA icv injection (iv, 15mg/kg) (fig. 12). It can be noted that the anti-CD 38 antibody daratumab (DARA, 15mg/kg iv) failed to demonstrate neuroprotective activity.

To see if the B08 anti-CD 38 antibody also protected neurons while the neurodegenerative process was in progress, we injected B08 anti-CD 38 antibody two days after 6-OHDAicv injection (at 15mg/kg iv). Even after the onset of denaturation, we observed strong neuroprotective activity of the B08 anti-CD 38 antibody (fig. 13). This demonstrates the strong neuroprotective effect of the B08 anti-CD 38 antibody.

In conclusion, it was demonstrated that the neuroprotective effect of the B08 and D06 anti-CD 38 antibodies was better than the efficacy of the neuroprotective effect obtained with HB7 anti-CD 38 antibody. It can be noted that this neuroprotective effect is still observed when the compound is injected intravenously at the time of the neurodegenerative process.

Example 5: in vitro anti-inflammatory properties of B08, C05, D06 and D07 anti-CD 38 antibodies

To determine whether agonistic anti-CD 38 antibodies (clones B08, C05, D06 and D07) have anti-inflammatory properties, we evaluated their effect on anti-inflammatory cytokine IL-10 release in vitro using human Peripheral Blood Mononuclear Cells (PBMCs) from 3 healthy donors. We observed that anti-CD 38 antibodies (clones B08, C05, D06 and D07) all statistically significantly increased IL-10 release, demonstrating anti-inflammatory properties (fig. 14). Importantly, HB7 failed to statistically significantly increase IL-10 release in human PBMC cultures in this assay, indicating that B08, C05, D06 and D07 anti-CD 38 antibodies have a higher anti-inflammatory effect than HB7 anti-CD 38 antibodies.

It was concluded that agonistic anti-CD 38 antibodies, represented by clones B08, C05, D06 and D07, increased IL-10 release from PBMCs in vitro. In addition, since IL-10 is involved in the general mechanism of inflammation and has been reported in many autoimmune diseases, agonistic anti-CD 38 antibodies represented by clones B08, C05, D06 and D07 provide a new therapeutic strategy for the treatment and/or prevention of inflammatory diseases and/or autoimmune diseases.

Example 6: neuroprotective effect of B08 anti-CD 38 antibody in Experimental Autoimmune Encephalomyelitis (EAE) mouse model of Multiple Sclerosis (MS), an Autoimmune neuroinflammatory disease

Multiple Sclerosis (MS) is a progressive inflammatory and demyelinating disease of the human Central Nervous System (CNS) in which the immune system is suspected to play an important role in the pathogenesis of this disease (Lassmann,2008.J Neurol sci.274(1-2): 45-7). EAE is a disease induced in mice that affects the central nervous system and constitutes a model for human MS. Following subcutaneous injection of an emulsion containing Complete Freund's Adjuvant (CFA) mixed with oligodendrocyte membrane proteins such as Myelin Oligodendrocyte Glycoprotein (MOG), the immune response starts from the periphery and causes inflammation in the CNS within 2 weeks. At the same time, we can observe progressive ascending paralysis (from tail to hind limb to forelimb). To determine whether the B08 anti-CD 38 antibody had a beneficial effect in this disease, we injected this antibody (15mg/kg, at the onset of symptoms (10 days after MOG injection) and on day 20). We observed that the B08 anti-CD 38 antibody significantly reduced clinical scores compared to PBS-injected mice (figure 15).

It was concluded that intravenous injection of an agonistic anti-CD 38 antibody (clone B08) at the onset of symptoms improved clinical scores in the EAE mouse model of autoimmune neuroinflammatory disease MS.

Example 7: anti-inflammatory properties of B08 agonistic anti-CD 38 antibody in vivo in mouse model of Dextran Sodium Sulfate (DSS) -induced colitis

To demonstrate the effect of B08 agonistic anti-CD 38 antibodies in a pure inflammatory model, we decided to test their effect in a mouse model of Dextran Sodium Sulfate (DSS) -induced colitis. Due to toxicity of DSS to colon epithelial cells, its administration can lead to human ulcerative colitis-like lesions, resulting in impaired mucosal barrier function. Clinical observations similar to human pathology, such as weight loss, diarrhea, and fecal occult blood in stool, are commonly observed in the DSS model. In addition, a reduction in colon length is also a feature of this model. Importantly, studies have shown that DSS colitis is primarily involved in the activation of lymphocytes, neutrophils and macrophages (Eichole and Kharbanda,2017.World J gastroenterol.23(33): 6016-29).

We observed that administration of B08 anti-DC 38 antibody (15mg/kg, iv) statistically significantly inhibited the reduction in colon length (fig. 16).

It was concluded that an agonistic anti-CD 38 antibody (clone B08) increased colon length in a DSS mouse model of inflammatory bowel disease.

Example 8: use of CD38 levels as biomarkers for therapeutic use of sCD31 or agonistic anti-CD 38 antibodies

We observed that sCD31 and some anti-CD 38 antibodies that reproduce binding of sCD31 to CD38 have neuroprotective effects. Thus, we evaluated whether measuring CD38 levels could be used to identify patients that would benefit from such anti-CD 38 therapy. We observed a significant increase in plasma CD38 levels in plasma samples from ALS patients compared to Healthy Controls (HC) (figure 17). This indicates that CD38 levels can be used as a marker for selecting patients for administration of the anti-CD 38 antibodies described herein.

Example 9: materials and methods for examples 1-7

1-in vitro experiments

1.1-midbrain cell culture

Animals were treated according to the guidelines of the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996), european instructions 86/609 and the guidelines of the local institutional animal Care and Use committee. Preparation of culture Medium from ventral midbrain (ventral mesencephalon) of Wistar rat embryos 15.5 gestational age (Janvier Breeding Center, Le Genest St Isle, France)And (5) nourishing the food. Dispersed cells (dissociated cells) in suspension obtained by mechanical milling of midbrain tissue fragments at 1.2-1.5x 10⁵Individual cell/cm²Is inoculated onto a tissue culture support precoated with 1mg/mL polyethyleneimine diluted in borate buffer at pH 8.3, as described (Michel et al, 1997.J neurohem.69 (4): 1499-507). The cultures were then maintained in N5 medium supplemented with 5mM glucose, 5% horse serum and 0.5% fetal bovine serum, excluding the first 3 DIVs, at a concentration of 2.5% fetal bovine serum, which facilitated initial maturation of the cultures (Guerreiro et al, 2008.Mol Pharmacol.74(4): 980-9). They were fed daily by replacing 70% of the medium. Typically, mesencephalon cultures are established on Nunc 24-well culture plates (Thermofischer Scientific, Rochester, N.Y.). Note that these cultures contained only dopaminergic Tyrosine Hydroxylase (TH)⁺Neurons (Traver et al, 2006.Mol Pharmacol.70(1): 30-40). TH⁺Neurons account for approximately 1-2% of the total number of neuronal cells present in these cultures. The survival of DA neurons was assessed by counting cells that were immunopositive for TH, as described (Toulorge et al, 2011. Faseeb J.25(8): 2563-73).

1.2-MPP⁺Poisoning model

MPP in culture⁺Treatment with K by prolonged exposure to depolarizing concentrations⁺(30mM) to prevent the spontaneous death process in the presence of the glutamate receptor antagonist MK801 (5. mu.M) which prevents unwanted excitotoxic damage as described previously (Douhou et al, 2001.J neurohem.78 (1): 163-74). MPP⁺And the treatment of potential neuroprotective molecules was performed between 5 and 7 DIV.

1.3 quantification of neuronal survival

The cultures were fixed for 12 minutes using 4% formaldehyde in Dulbecco's phosphate-buffered saline (PBS), then washed twice with PBS, and then incubated with the following antibodies at 4 ℃ for 24 hours. DA neuron survival was assessed using 1/5000 diluted monoclonal anti-TH antibody (ImmunoStar, inc., Hudson, WI). Antibodies were diluted in PBS containing 0.2% Triton X-100. Primary antibodies were detected using an Alexa Fluor-488 conjugate of an anti-mouse IgG antibody.

1.4 human PBMC cell culture

3 healthy volunteers (donor 1: female, 23 years old; donor 2: male, 29 years old; donor 3: female, 20 years old) served as blood donors. Venous blood (5mL) was collected into heparinized tubes (heparinized tubes) and immediately processed. Briefly, blood 1/1 was diluted in pre-warmed RPMI 1640 medium. PBMCs were separated using gradient centrifugation in Ficoll solution (Sigma; 300x g, 30 min). The isolated PBMC were resuspended in 1 volume of pre-warmed RPMI 1640 medium and centrifuged again (300x g, 30 min). PBMC were treated with 10⁷The concentration of individual cells/ml was resuspended in RPMI 1640 medium supplemented with 10% human serum (Sigma-Aldrich). Cells were seeded into 96-well microplates (100. mu.L per well). Immediately after inoculation, test compound (10. mu.g/mL), LPS (500ng/mL) and interferon-. gamma.1B (100ng/mL) were added to PBMC cultures. After 24 hours, the cell culture supernatant was collected and frozen at-80 ℃ for further processing.

1.5-IL-10ELISA assay

ELISA human IL-10 kit was used according to manufacturer's instructionsIL-10 levels in cell culture supernatants were determined.

1.6 statistical analysis

A simple comparison between the two groups was performed using student's t-test. Multiple comparisons were performed on a single reference group by one-way anova followed by Dunnett's test (if possible). When full pairwise comparisons (all pair comparisons) are required, Student-Newman-Keuls test is used. S.e.m. values were from at least three independent experiments.

2-in vivo experiments

2.1-animals

Animals were treated according to the guidelines for experimental animal care and use (national research committee, 1996), european directive 2010/63/EU and the guidelines of the animal care and use committee of the local institution. For all studies, 8 to 10 week old male C57Bl/6 mice (Janvier, france) were used. They were kept in a 12:12h light/dark cycle (light on 8 early). The room temperature was maintained at 20 ℃ and standard diet and tap water were freely available.

2.2-6-OHDA and MPTP mouse models

a) Design of experiments

Two in vivo mouse models were used: 6-OHDA mouse model and MPTP mouse model. For the 6-OHDA mouse model, the intoxication protocol was based on a unilateral stereotactic intrastriatal injection of 6-OHDA into the right striatum. Treatment by intracerebral or intravenous injection is performed one day prior to the surgical stereotactic procedure (surgical stereotactic procedure), simultaneously with 6-OHDA during the surgical stereotactic procedure, or two days after the injection of 6-OHDA. Animals were sacrificed 8 days after the surgical stereotactic procedure.

For the MPTP mouse model, 4 MPTP (20mg/kg) injections were performed. Intravenous injection of anti-CD 38 clone HB7 antibody was performed 2 days before MPTP injection. Mice were sacrificed 7 days after MPTP injection.

b) Surgical stereotactic procedure

For the unilateral 6-OHDA mouse model, animals were anesthetized with chloral hydrate (400mg/kg, i.p.) and placed in a stereotactic frame suitable for mice. Injections were performed using a Hamilton syringe at the following coordinates: AP: +0.85cm, ML: ± 2cm, DV: 3.4cm (corresponding to the atlas (atlas) of Franklin and Paxinos, 1997). A total volume of 2.5. mu.L with or without 5. mu.g of 6-OHDA diluted in PBS was injected in the presence or absence of test treatment. The needle was left in place for 10 minutes after injection and then withdrawn.

c) Tissue preparation

Mice were sacrificed by cervical dislocation and heads were separated to collect brain and blood samples. The brain was fixed in paraformaldehyde solution (4%) for 1 day, then soaked in sucrose (30%) for 2 days, then frozen at-80 ℃ for further analysis.

d) Brain section, immunofluorescence and neuronal count (enumeration)

Each brain was sectioned using a cryo-microtome at-30 ℃. The thickness of each slice was 20 μm. Sections were taken around substantia nigra (substantia nigra) (80 sections from 51 to 63 according to Allen Mouse Brain (Allen Mouse Brain).

The sections were then washed in PBS and incubated with anti-tyrosine hydroxylase with agitation for 2 days at 4 ℃ to convert dopaminergic neuronsAnd (6) dyeing. Sections were mounted on DAPI (Sigma)) The nuclei were stained with the corresponding secondary antibody by incubation at room temperature for 2 hours in the presence and fixed on gelatin-coated slides.

Sections were imaged using Nikon TE2000U equipped with a Hamamatsu ORCE-ER camera or Zeiss Axio vert.a1 equipped with an axiocam 503 monochrome camera. Using Image J software or ZenImage analysis is performed.

2.3-IL-10ELISA assay

Will be collected in a collecting tube (500EDTA-K3, SARSTEDT) were centrifuged at 3000 rpm for 15 minutes. The supernatant (plasma) was collected and transferred to a test tube and frozen at-80 ℃ for further analysis. ELISA IL-10 kit was used according to manufacturer's instructionsIL-10 levels in plasma samples were determined.

2.4-EAE mouse model

a) Design of experiments

Animals were anesthetized by isoflurane inhalation (15 min). On day 0, mice were immunized by subcutaneous injection of an emulsion containing 100 μ g of MOG peptide 35-55 and 500 μ g of HKMT (Heat-inactivated Mycobacterium tuberculosis) in complete Freund's adjuvant. Two hours later, animals received intraperitoneal injections of 500ng of pertussis toxin on days 0 and 2. Test compounds were injected intravenously on days 10 and 20 after subcutaneous injection of the emulsion. Animals were sacrificed on day 28.

b) Clinical score assessment

Following immunization, animals were monitored daily for body weight and clinical symptoms. Clinical scores were graded according to the following scale: 0: no clinical symptoms; 0.5: partial soft tail; 1: paralysis of the tail, normal gait; 1.5: paralysis of the tail, uncoordinated movement, and paresis of hind limbs; 2: paralysis of hind limbs or no reaction to squeeze and pinch; 2.5: one hind limb is paralyzed and the other is weak; 3: paralysis of both hind limbs; 3.5: paralysis of both hind limbs and weakness of the forelimbs; 4: a moribund state.

2.5-DSS mouse model

a) Design of experiments

Experimental colitis was induced in mice by free provision of 3% (w/v) DSS-containing drinking water for 5 days, while control mice received tap water only. Test compound (15mg/kg) was administered intravenously when DSS treatment began. Mice were sacrificed 9 days after the start of DSS treatment.

b) Statistical analysis

Statistical analysis and mapping were performed using Sigma Plot software. Different measurements (if applicable) can be made based on one-way or two-way ANOVA and post hoc test (post hoc test) data.

Sequence listing

<110> ENCEFA company (ENCEFA)

L-Blassam (BRESSAC Laurence)

S, Gracilo (GUERREIRO Serge)

D & Charogee (TOULORGE Damien)

<120> CD31 competitors and uses thereof

<130> CV - 1204/PCT

<150> EP19305086.1

<151> 2019-01-23

<160> 43

<170> BiSSAP 1.3.6

<210> 1

<211> 738

<212> PRT

<213> Intelligent (Homo sapiens)

<220>

<223> CD31

<400> 1

Met Gln Pro Arg Trp Ala Gln Gly Ala Thr Met Trp Leu Gly Val Leu

1 5 10 15

Leu Thr Leu Leu Leu Cys Ser Ser Leu Glu Gly Gln Glu Asn Ser Phe

20 25 30

Thr Ile Asn Ser Val Asp Met Lys Ser Leu Pro Asp Trp Thr Val Gln

35 40 45

Asn Gly Lys Asn Leu Thr Leu Gln Cys Phe Ala Asp Val Ser Thr Thr

50 55 60

Ser His Val Lys Pro Gln His Gln Met Leu Phe Tyr Lys Asp Asp Val

65 70 75 80

Leu Phe Tyr Asn Ile Ser Ser Met Lys Ser Thr Glu Ser Tyr Phe Ile

85 90 95

Pro Glu Val Arg Ile Tyr Asp Ser Gly Thr Tyr Lys Cys Thr Val Ile

100 105 110

Val Asn Asn Lys Glu Lys Thr Thr Ala Glu Tyr Gln Val Leu Val Glu

115 120 125

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

130 135 140

Gly Gly Ile Val Arg Val Asn Cys Ser Val Pro Glu Glu Lys Ala Pro

145 150 155 160

Ile His Phe Thr Ile Glu Lys Leu Glu Leu Asn Glu Lys Met Val Lys

165 170 175

Leu Lys Arg Glu Lys Asn Ser Arg Asp Gln Asn Phe Val Ile Leu Glu

180 185 190

Phe Pro Val Glu Glu Gln Asp Arg Val Leu Ser Phe Arg Cys Gln Ala

195 200 205

Arg Ile Ile Ser Gly Ile His Met Gln Thr Ser Glu Ser Thr Lys Ser

210 215 220

Glu Leu Val Thr Val Thr Glu Ser Phe Ser Thr Pro Lys Phe His Ile

225 230 235 240

Ser Pro Thr Gly Met Ile Met Glu Gly Ala Gln Leu His Ile Lys Cys

245 250 255

Thr Ile Gln Val Thr His Leu Ala Gln Glu Phe Pro Glu Ile Ile Ile

260 265 270

Gln Lys Asp Lys Ala Ile Val Ala His Asn Arg His Gly Asn Lys Ala

275 280 285

Val Tyr Ser Val Met Ala Met Val Glu His Ser Gly Asn Tyr Thr Cys

290 295 300

Lys Val Glu Ser Ser Arg Ile Ser Lys Val Ser Ser Ile Val Val Asn

305 310 315 320

Ile Thr Glu Leu Phe Ser Lys Pro Glu Leu Glu Ser Ser Phe Thr His

325 330 335

Leu Asp Gln Gly Glu Arg Leu Asn Leu Ser Cys Ser Ile Pro Gly Ala

340 345 350

Pro Pro Ala Asn Phe Thr Ile Gln Lys Glu Asp Thr Ile Val Ser Gln

355 360 365

Thr Gln Asp Phe Thr Lys Ile Ala Ser Lys Ser Asp Ser Gly Thr Tyr

370 375 380

Ile Cys Thr Ala Gly Ile Asp Lys Val Val Lys Lys Ser Asn Thr Val

385 390 395 400

Gln Ile Val Val Cys Glu Met Leu Ser Gln Pro Arg Ile Ser Tyr Asp

405 410 415

Ala Gln Phe Glu Val Ile Lys Gly Gln Thr Ile Glu Val Arg Cys Glu

420 425 430

Ser Ile Ser Gly Thr Leu Pro Ile Ser Tyr Gln Leu Leu Lys Thr Ser

435 440 445

Lys Val Leu Glu Asn Ser Thr Lys Asn Ser Asn Asp Pro Ala Val Phe

450 455 460

Lys Asp Asn Pro Thr Glu Asp Val Glu Tyr Gln Cys Val Ala Asp Asn

465 470 475 480

Cys His Ser His Ala Lys Met Leu Ser Glu Val Leu Arg Val Lys Val

485 490 495

Ile Ala Pro Val Asp Glu Val Gln Ile Ser Ile Leu Ser Ser Lys Val

500 505 510

Val Glu Ser Gly Glu Asp Ile Val Leu Gln Cys Ala Val Asn Glu Gly

515 520 525

Ser Gly Pro Ile Thr Tyr Lys Phe Tyr Arg Glu Lys Glu Gly Lys Pro

530 535 540

Phe Tyr Gln Met Thr Ser Asn Ala Thr Gln Ala Phe Trp Thr Lys Gln

545 550 555 560

Lys Ala Ser Lys Glu Gln Glu Gly Glu Tyr Tyr Cys Thr Ala Phe Asn

565 570 575

Arg Ala Asn His Ala Ser Ser Val Pro Arg Ser Lys Ile Leu Thr Val

580 585 590

Arg Val Ile Leu Ala Pro Trp Lys Lys Gly Leu Ile Ala Val Val Ile

595 600 605

Ile Gly Val Ile Ile Ala Leu Leu Ile Ile Ala Ala Lys Cys Tyr Phe

610 615 620

Leu Arg Lys Ala Lys Ala Lys Gln Met Pro Val Glu Met Ser Arg Pro

625 630 635 640

Ala Val Pro Leu Leu Asn Ser Asn Asn Glu Lys Met Ser Asp Pro Asn

645 650 655

Met Glu Ala Asn Ser His Tyr Gly His Asn Asp Asp Val Arg Asn His

660 665 670

Ala Met Lys Pro Ile Asn Asp Asn Lys Glu Pro Leu Asn Ser Asp Val

675 680 685

Gln Tyr Thr Glu Val Gln Val Ser Ser Ala Glu Ser His Lys Asp Leu

690 695 700

Gly Lys Lys Asp Thr Glu Thr Val Tyr Ser Glu Val Arg Lys Ala Val

705 710 715 720

Pro Asp Ala Val Glu Ser Arg Tyr Ser Arg Thr Glu Gly Ser Leu Asp

725 730 735

Gly Thr

<210> 2

<211> 300

<212> PRT

<213> Intelligent (Homo sapiens)

<220>

<223> CD38

<400> 2

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> 3

<211> 585

<212> PRT

<213> little mouse (Mus musculus)

<220>

<223> soluble CD31(sCD31)

<400> 3

Met Leu Leu Ala Leu Gly Leu Thr Leu Val Leu Tyr Ala Ser Leu Gln

1 5 10 15

Ala Glu Glu Asn Ser Phe Thr Ile Asn Ser Ile His Met Glu Ser Leu

20 25 30

Pro Ser Trp Glu Val Met Asn Gly Gln Gln Leu Thr Leu Glu Cys Leu

35 40 45

Val Asp Ile Ser Thr Thr Ser Lys Ser Arg Ser Gln His Arg Val Leu

50 55 60

Phe Tyr Lys Asp Asp Ala Met Val Tyr Asn Val Thr Ser Arg Glu His

65 70 75 80

Thr Glu Ser Tyr Val Ile Pro Gln Ala Arg Val Phe His Ser Gly Lys

85 90 95

Tyr Lys Cys Thr Val Met Leu Asn Asn Lys Glu Lys Thr Thr Ile Glu

100 105 110

Tyr Glu Val Lys Val His Gly Val Ser Lys Pro Lys Val Thr Leu Asp

115 120 125

Lys Lys Glu Val Thr Glu Gly Gly Val Val Thr Val Asn Cys Ser Leu

130 135 140

Gln Glu Glu Lys Pro Pro Ile Phe Phe Lys Ile Glu Lys Leu Glu Val

145 150 155 160

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

165 170 175

Phe Val Leu Met Glu Phe Pro Ile Glu Ala Gln Asp His Val Leu Val

180 185 190

Phe Arg Cys Gln Ala Gly Ile Leu Ser Gly Phe Lys Leu Gln Glu Ser

195 200 205

Glu Pro Ile Arg Ser Glu Tyr Val Thr Val Gln Glu Ser Phe Ser Thr

210 215 220

Pro Lys Phe Glu Ile Lys Pro Pro Gly Met Ile Ile Glu Gly Asp Gln

225 230 235 240

Leu His Ile Arg Cys Ile Val Gln Val Thr His Leu Val Gln Glu Phe

245 250 255

Thr Glu Ile Ile Ile Gln Lys Asp Lys Ala Ile Val Ala Thr Ser Lys

260 265 270

Gln Ser Ser Glu Ala Val Tyr Ser Val Met Ala Met Val Glu Tyr Ser

275 280 285

Gly His Tyr Thr Cys Lys Val Glu Ser Asn Arg Ile Ser Lys Ala Ser

290 295 300

Ser Ile Met Val Asn Ile Thr Glu Leu Phe Pro Lys Pro Lys Leu Glu

305 310 315 320

Phe Ser Ser Ser Arg Leu Asp Gln Gly Glu Leu Leu Asp Leu Ser Cys

325 330 335

Ser Val Ser Gly Thr Pro Val Ala Asn Phe Thr Ile Gln Lys Glu Glu

340 345 350

Thr Val Leu Ser Gln Tyr Gln Asn Phe Ser Lys Ile Ala Glu Glu Ser

355 360 365

Asp Ser Gly Glu Tyr Ser Cys Thr Ala Gly Ile Gly Lys Val Val Lys

370 375 380

Arg Ser Gly Leu Val Pro Ile Gln Val Cys Glu Met Leu Ser Lys Pro

385 390 395 400

Ser Ile Phe His Asp Ala Lys Ser Glu Ile Ile Lys Gly His Ala Ile

405 410 415

Gly Ile Ser Cys Gln Ser Glu Asn Gly Thr Ala Pro Ile Thr Tyr His

420 425 430

Leu Met Lys Ala Lys Ser Asp Phe Gln Thr Leu Glu Val Thr Ser Asn

435 440 445

Asp Pro Ala Thr Phe Thr Asp Lys Pro Thr Arg Asp Met Glu Tyr Gln

450 455 460

Cys Arg Ala Asp Asn Cys His Ser His Pro Ala Val Phe Ser Glu Ile

465 470 475 480

Leu Arg Val Arg Val Ile Ala Pro Val Asp Glu Val Val Ile Ser Ile

485 490 495

Leu Ser Ser Asn Glu Val Gln Ser Gly Ser Glu Met Val Leu Arg Cys

500 505 510

Ser Val Lys Glu Gly Thr Ser Pro Ile Thr Phe Gln Phe Tyr Lys Glu

515 520 525

Lys Glu Asp Arg Pro Phe His Gln Ala Val Val Asn Asp Thr Gln Ala

530 535 540

Phe Trp His Asn Lys Gln Ala Ser Lys Lys Gln Glu Gly Gln Tyr Tyr

545 550 555 560

Cys Thr Ala Ser Asn Arg Ala Ser Ser Met Arg Thr Ser Pro Arg Ser

565 570 575

Ser Thr Leu Ala Val Arg Val Phe Leu

580 585

<210> 4

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> VH-CDR 1-consensus

<220>

<221> SITE (SITE)

<222> 7

<223> X is selected from Tyr (Y), Asn (N) and Ser (S)

<400> 4

Gly Phe Thr Phe Ser Asn Xaa

1 5

<210> 5

<211> 6

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> SITE (SITE)

<222> 1

<223> X is selected from Ser (S) and Tyr (Y)

<220>

<223> VH-CDR 2-consensus

<220>

<221> SITE (SITE)

<222> 6

<223> X is selected from Tyr (Y), Asp (D), Asn (N) and Ser (S)

<400> 5

Xaa Gly Ser Ser Arg Xaa

1 5

<210> 6

<211> 14

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> SITE (SITE)

<222> 1

<223> X is selected from Ser (S) and a vacancy

<220>

<223> VH-CDR 3-consensus

<220>

<221> SITE (SITE)

<222> 2

<223> X is selected from Ser (S) and a vacancy

<220>

<221> SITE (SITE)

<222> 3

<223> X is selected from Ser (S) and Tyr (Y)

<220>

<221> SITE (SITE)

<222> 4

<223> X is selected from Ser (S) and Asp (D)

<220>

<221> SITE (SITE)

<222> 5

<223> X is selected from Tyr (Y), Ser (S), Asp (D) and Gly (G)

<220>

<221> SITE (SITE)

<222> 7

<223> X is selected from Tyr (Y) and Gly (G)

<220>

<221> SITE (SITE)

<222> 8

<223> X is selected from Ser (S), Tyr (Y) and Phe (F)

<220>

<221> SITE (SITE)

<222> 9

<223> X is selected from Gly (G) and Asp (D)

<220>

<221> SITE (SITE)

<222> 10

<223> X is selected from Asn (N), Tyr (Y) and Ser (S)

<400> 6

Xaa Xaa Xaa Xaa Xaa Tyr Xaa Xaa Xaa Xaa Gly Met Asp Val

1 5 10

<210> 7

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> VH-CDR1

<400> 7

Gly Phe Thr Phe Ser Asn Tyr

1 5

<210> 8

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> VH-CDR1

<400> 8

Gly Phe Thr Phe Ser Asn Asn

1 5

<210> 9

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> VH-CDR1

<400> 9

Gly Phe Thr Phe Ser Asn Ser

1 5

<210> 10

<211> 6

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> VH-CDR2

<400> 10

Ser Gly Ser Ser Arg Ser

1 5

<210> 11

<211> 6

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> VH-CDR2

<400> 11

Ser Gly Ser Ser Arg Tyr

1 5

<210> 12

<211> 6

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> VH-CDR2

<400> 12

Ser Gly Ser Ser Arg Asn

1 5

<210> 13

<211> 6

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> VH-CDR2

<400> 13

Tyr Gly Ser Ser Arg Asp

1 5

<210> 14

<211> 6

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> VH-CDR2

<400> 14

Ser Gly Ser Ser Arg Asp

1 5

<210> 15

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> VH-CDR3

<400> 15

Ser Ser Asp Asp Tyr Tyr Tyr Asp Tyr Gly Met Asp Val

1 5 10

<210> 16

<211> 14

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> VH-CDR3

<400> 16

Ser Ser Ser Ser Tyr Tyr Tyr Ser Gly Asn Gly Met Asp Val

1 5 10

<210> 17

<211> 14

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> VH-CDR3

<400> 17

Ser Ser Tyr Ser Ser Tyr Gly Ser Gly Asn Gly Met Asp Val

1 5 10

<210> 18

<400> 18

000

<210> 19

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> VH-CDR3

<400> 19

Ser Ser Gly Tyr Tyr Phe Gly Tyr Gly Met Asp Val

1 5 10

<210> 20

<211> 14

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> VH-CDR3

<400> 20

Ser Ser Ser Ser Tyr Tyr Tyr Ser Gly Ser Gly Met Asp Val

1 5 10

<210> 21

<211> 14

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> VL-CDR 1-consensus

<220>

<221> SITE (SITE)

<222> 10

<223> X is selected from Ser (S) and Asn (N)

<220>

<221> SITE (SITE)

<222> 11

<223> X is selected from Ser (S) and Tyr (Y)

<220>

<221> SITE (SITE)

<222> 12

<223> X is selected from Tyr (Y), and Ser (S)

<400> 21

Ala Gly Thr Ser Ser Asp Val Gly Gly Xaa Xaa Xaa Val Ser

1 5 10

<210> 22

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> SITE (SITE)

<222> 1

<223> X is selected from Tyr (Y), Ser (S) and Asp (D)

<220>

<223> VL-CDR 2-consensus

<220>

<221> SITE (SITE)

<222> 4

<223> X is selected from Tyr (Y) and Asn (N)

<400> 22

Xaa Asp Ser Xaa Arg Pro Ser

1 5

<210> 23

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> VL-CDR 3-consensus

<400> 23

Ser Thr Arg Val Phe Gly Gly Gly Thr

1 5

<210> 24

<211> 14

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> VL-CDR1

<400> 24

Ala Gly Thr Ser Ser Asp Val Gly Gly Ser Tyr Ser Val Ser

1 5 10

<210> 25

<211> 14

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> VL-CDR1

<400> 25

Ala Gly Thr Ser Ser Asp Val Gly Gly Ser Ser Tyr Val Ser

1 5 10

<210> 26

<400> 26

000

<210> 27

<211> 14

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> VL-CDR1

<400> 27

Ala Gly Thr Ser Ser Asp Val Gly Gly Ser Tyr Tyr Val Ser

1 5 10

<210> 28

<211> 14

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> VL-CDR1

<400> 28

Ala Gly Thr Ser Ser Asp Val Gly Gly Asn Ser Tyr Val Ser

1 5 10

<210> 29

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> VL-CDR2

<400> 29

Asp Asp Ser Asn Arg Pro Ser

1 5

<210> 30

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> VL-CDR2

<400> 30

Tyr Asp Ser Tyr Arg Pro Ser

1 5

<210> 31

<400> 31

000

<210> 32

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> VL-CDR2

<400> 32

Ser Asp Ser Tyr Arg Pro Ser

1 5

<210> 33

<211> 122

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> HCVR B08

<400> 33

Glu Val Gln Leu Val Glu Ser Gly Gly Ser Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Ser Ser Ile Ser Gly Ser Ser Arg Ser Ile Tyr Tyr Ala Asp Phe Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

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

85 90 95

Val Arg Ser Ser Asp Asp Tyr Tyr Tyr Asp Tyr Gly Met Asp Val Trp

100 105 110

Gly Arg Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 34

<211> 123

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> HCVR C05

<400> 34

Glu Val Gln Leu Val Glu Ser Gly Gly Ser Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Asn

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Ser Ser Ile Ser Gly Ser Ser Arg Tyr Ile Ser Tyr Ala Asp Phe Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

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

85 90 95

Val Arg Ser Ser Ser Ser Tyr Tyr Tyr Ser Gly Asn Gly Met Asp Val

100 105 110

Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 35

<211> 123

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> HCVR C06

<400> 35

Glu Val Gln Leu Val Glu Ser Gly Gly Ser Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ser

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Ser Ser Ile Ser Gly Ser Ser Arg Asn Ile Tyr Tyr Ala Asp Phe Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

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

85 90 95

Val Arg Ser Ser Tyr Ser Ser Tyr Gly Ser Gly Asn Gly Met Asp Val

100 105 110

Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 36

<211> 121

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> HCVR D06

<400> 36

Glu Val Gln Leu Val Glu Ser Gly Gly Ser Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr

20 25 30

Asp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Ser Tyr Ile Tyr Gly Ser Ser Arg Asp Ile Ser Tyr Ala Asp Phe Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

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

85 90 95

Val Arg Ser Ser Gly Tyr Tyr Phe Gly Tyr Gly Met Asp Val Trp Gly

100 105 110

Arg Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 37

<211> 123

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> HCVR D07

<400> 37

Glu Val Gln Leu Val Glu Ser Gly Gly Ser Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ser

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Ser Tyr Ile Ser Gly Ser Ser Arg Asp Ile Ser Tyr Ala Asp Phe Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

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

85 90 95

Val Arg Ser Ser Ser Ser Tyr Tyr Tyr Ser Gly Ser Gly Met Asp Val

100 105 110

Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 38

<211> 123

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> HCVR D10

<400> 38

Glu Val Gln Leu Val Glu Ser Gly Gly Ser Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ser

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Ser Ser Ile Ser Gly Ser Ser Arg Asp Ile Tyr Tyr Ala Asp Phe Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

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

85 90 95

Val Arg Ser Ser Ser Ser Tyr Tyr Tyr Ser Gly Asn Gly Met Asp Val

100 105 110

Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 39

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> LCVR B08

<400> 39

Gln Ser Val Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln

1 5 10 15

Ser Ile Thr Ile Ser Cys Ala Gly Thr Ser Ser Asp Val Gly Gly Ser

20 25 30

Tyr Ser Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Asp Asp Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu

65 70 75 80

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

85 90 95

Ser Thr Arg Val Phe Gly Gly Gly Thr Lys Leu

100 105

<210> 40

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> LCVR C05/C06

<400> 40

Gln Ser Val Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln

1 5 10 15

Ser Ile Thr Ile Ser Cys Ala Gly Thr Ser Ser Asp Val Gly Gly Ser

20 25 30

Ser Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Tyr Asp Ser Tyr Arg Pro Ser Gly Val Ser Asn Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Asn Thr Tyr Tyr

85 90 95

Ser Thr Arg Val Phe Gly Gly Gly Thr Lys Leu

100 105

<210> 41

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> LCVR D06

<400> 41

Gln Ser Val Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln

1 5 10 15

Ser Ile Thr Ile Ser Cys Ala Gly Thr Ser Ser Asp Val Gly Gly Ser

20 25 30

Tyr Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Ser Asp Ser Tyr Arg Pro Ser Gly Val Ser Asn Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Asn Thr Ala Tyr

85 90 95

Ser Thr Arg Val Phe Gly Gly Gly Thr Lys Leu

100 105

<210> 42

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> LCVR D07/D10

<400> 42

Gln Ser Val Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln

1 5 10 15

Ser Ile Thr Ile Ser Cys Ala Gly Thr Ser Ser Asp Val Gly Gly Asn

20 25 30

Ser Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Tyr Asp Ser Tyr Arg Pro Ser Gly Val Ser Asn Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Asn Thr Tyr Tyr

85 90 95

Ser Thr Arg Val Phe Gly Gly Gly Thr Lys Leu

100 105

<210> 43

<211> 609

<212> PRT

<213> Intelligent (Homo sapiens)

<220>

<223> serum albumin

<400> 43

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala

1 5 10 15

Tyr Ser Arg Gly Val Phe Arg Arg Asp Ala His Lys Ser Glu Val Ala

20 25 30

His Arg Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu

35 40 45

Ile Ala Phe Ala Gln Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val

50 55 60

Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp

65 70 75 80

Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp

85 90 95

Lys Leu Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala

100 105 110

Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln

115 120 125

His Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val

130 135 140

Asp Val Met Cys Thr Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys

145 150 155 160

Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro

165 170 175

Glu Leu Leu Phe Phe Ala Lys Arg Tyr Lys Ala Ala Phe Thr Glu Cys

180 185 190

Cys Gln Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu

195 200 205

Leu Arg Asp Glu Gly Lys Ala Ser Ser Ala Lys Gln Arg Leu Lys Cys

210 215 220

Ala Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val

225 230 235 240

Ala Arg Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser

245 250 255

Lys Leu Val Thr Asp Leu Thr Lys Val His Thr Glu Cys Cys His Gly

260 265 270

Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile

275 280 285

Cys Glu Asn Gln Asp Ser Ile Ser Ser Lys Leu Lys Glu Cys Cys Glu

290 295 300

Lys Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp

305 310 315 320

Glu Met Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser

325 330 335

Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly

340 345 350

Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val

355 360 365

Leu Leu Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys

370 375 380

Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu

385 390 395 400

Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys

405 410 415

Glu Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu

420 425 430

Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val

435 440 445

Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His

450 455 460

Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val

465 470 475 480

Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg

485 490 495

Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe

500 505 510

Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala

515 520 525

Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu

530 535 540

Arg Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys

545 550 555 560

Pro Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala

565 570 575

Ala Phe Val Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe

580 585 590

Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly

595 600 605

Leu

Detailed Description

The present invention relates to compounds that specifically compete with CD31 for binding to CD 38.

In one embodiment, the compounds of the invention compete with the extracellular domain of CD 31. In one embodiment, the compounds of the invention compete with Ig-like domains 1-3 of the extracellular domain of CD 31.

SEQ ID NO:1 the amino acid sequence of human CD31 comprising the following features: a signal peptide of amino acids 1 to 27; ig-like domain 1 of amino acids 34 to 121; ig-like domain 2 of amino acids 145 to 233; ig-like domain 3 of amino acids 236 to 315; ig-like domain 4 of amino acids 328 to 401; ig-like domain 5 of amino acids 424 to 493; ig-like domain 6 of amino acids 499 to 591; a membrane-proximal domain of amino acids 592 to 601 (juxta-membrane domain); a transmembrane domain of amino acids 602 to 620 and a cytoplasmic domain of amino acids 621 to 738.

Thus, in one embodiment, the compounds of the invention are conjugated to the extracellular domain of human CD31, preferably to the amino acid sequence of SEQ ID NO:1 from amino acids 1 to 601 or 28 to 601. In one embodiment, the compounds of the present invention are conjugated to Ig-like domains 1-3 of human CD31, preferably to the amino acid sequence of SEQ ID NO:1 from amino acids 1 to 315 or 28 to 315 or 1 to 327 or 28 to 327.

In one embodiment, the compound of the invention is selected from the group comprising or consisting of: peptides (including recombinant or chimeric peptides), antibodies, antigen-binding fragments thereof, antigen-binding antibody mimetics, oligonucleotides, and small organic molecules.

In one embodiment, the compounds of the invention specifically bind to CD38, preferably human CD 38.

The specific binding between the compounds of the present invention and CD38 means that the compounds exhibit considerable affinity for CD 38.

The affinity of the compounds of the present invention for CD38 can be determined by various methods known to those skilled in the art. These methods include, but are not limited to, biosensor analysis (including, for example, Biacore analysis), Blitz analysis, and Scatchard plots.

Alternatively or additionally, it can be readily detected whether a compound of the invention binds to CD38 by (among other things) comparing the reaction of the compound with CD38 or a fragment thereof, in particular a fragment comprising an epitope of CD38 or consisting of an epitope of CD38, and the reaction of the compound with a protein or antigen other than CD38 or a fragment thereof.

In one embodiment where the compound of the invention is an antibody, antigen-binding fragment thereof, or antigen-binding antibody mimetic, "substantial affinity" may be defined as K_DAn affinity constant of about 10^-7M, preferably about 10^-8M or stronger. Specifically, when K_DAn affinity constant of about 10^-7M to about 10^-12M, preferably about 10^-8M to about 10^-12M, more preferably about 10^-9M to about 10^- ¹¹In the range of M, especially K_DAn affinity constant of about 10^-10M, the binding between an antibody, antigen-binding fragment thereof, or antigen-binding antibody mimetic described herein and CD38 is considered specific.

In one embodiment where the compound of the invention is an antibody, antigen-binding fragment thereof, or antigen-binding antibody mimetic, the compound has less than or equal to about 10 for CD38^-7M, preferably less than or equal to about 10^-8M, more preferably less than or equal to about 10^-9M, even more preferably less than or equal to about 10^-10K of M_D-an affinity constant; preferably, it can be determined by biosensor analysis, in particular by Biacore analysis.

In one embodiment where the compounds of the invention are small organic molecules, "appreciable affinity" may applyIs defined as K_DAn affinity constant of about 10^-6And M. Specifically, when K_DAn affinity constant of about 10^-6M to about 10^-10M, preferably about 10^-7M to about 10^-9In the range of M, especially K_DAn affinity constant of about 10^-8M, the binding between the small organic molecules described herein and CD38 is considered specific; preferably, it can be determined by biosensor analysis, in particular by Biacore analysis.

In one embodiment where the compound of the invention is an oligonucleotide, "appreciable affinity" may be defined as K_DAn affinity constant of about 200nM or stronger. Specifically, when K_DAffinity constants in the range from about 10nM to about 200nM, preferably from about 50nM to about 150nM, especially K_DThe binding between the oligonucleotide according to the invention and CD38 is considered specific with an affinity constant of about 100 nM; preferably, it can be determined by biosensor analysis, in particular by Biacore analysis.

In one embodiment, the compounds of the invention specifically bind to at least one epitope within CD 38.

In one embodiment, the compounds of the invention specifically bind to at least one epitope within human CD 38.

In one embodiment, the epitope is linear. In one embodiment, the epitope is conformational.

In one embodiment, the epitope comprises 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6,5, 4, 3, 2 contiguous amino acids or non-contiguous residues.

In one embodiment, the epitope extends at least across a region having SEQ ID NO:2 from amino acid residue 1 to amino acid residue 300 of human CD38, or extends across the corresponding amino acid residues in homologous CD38 from other species.

In one embodiment, the epitope extends at least across a region having SEQ ID NO:2 from amino acid residue 1 to amino acid residue 296 of human CD38, or extends across the corresponding amino acid residues in homologous CD38 from other species.

In one embodiment, the epitope extends at least across a region having SEQ ID NO:2 from amino acid residue 1 to amino acid residue 294 of human CD38, or extends across the corresponding amino acid residues in homologous CD38 from other species.

In one embodiment, the epitope extends at least across a region having SEQ ID NO:2 from amino acid residue 1 to amino acid residue 285 of human CD38, or extends across the corresponding amino acid residues in homologous CD38 from other species.

In one embodiment, the epitope extends at least across a region having SEQ ID NO:2 from amino acid residue 70 to amino acid residue 300 of human CD38, or extends across the corresponding amino acid residues in homologous CD38 from other species.

In one embodiment, the epitope extends at least across a region having SEQ ID NO:2 from amino acid residue 70 to amino acid residue 296 of human CD38, or extends across the corresponding amino acid residues in homologous CD38 from other species.

In one embodiment, the epitope extends at least across a region having SEQ ID NO:2 from amino acid residue 70 to amino acid residue 294 of human CD38, or extends across the corresponding amino acid residues in homologous CD38 from other species.

In one embodiment, the epitope extends at least across a region having SEQ ID NO:2 from amino acid residue 70 to amino acid residue 285 of human CD38, or extends across the corresponding amino acid residues in homologous CD38 from other species.

In one embodiment, the epitope extends at least across a region having SEQ ID NO:2 from amino acid residue 189 to amino acid residue 300 of human CD38, or extends across the corresponding amino acid residues in homologous CD38 from other species.

In one embodiment, the epitope extends at least across a region having SEQ ID NO:2 from amino acid residue 189 to amino acid residue 296 of human CD38, or extends across the corresponding amino acid residues in homologous CD38 from other species.

In one embodiment, the epitope extends at least across a region having SEQ ID NO:2 from amino acid residue 189 to amino acid residue 294 of human CD38, or extends across the corresponding amino acid residues in homologous CD38 from other species.

In one embodiment, the epitope extends at least across a region having SEQ ID NO:2 from amino acid residue 189 to amino acid residue 285 of human CD38, or extends across the corresponding amino acid residues in homologous CD38 from other species.

In one embodiment, the epitope extends at least across a region having SEQ ID NO:2 from amino acid residue 219 to amino acid residue 300 of human CD38, or extends across the corresponding amino acid residues in homologous CD38 from other species.

In one embodiment, the epitope extends at least across a region having SEQ ID NO:2 from amino acid residue 219 to amino acid residue 296 of human CD38, or extends across the corresponding amino acid residues in homologous CD38 from other species.

In one embodiment, the epitope extends at least across a region having SEQ ID NO:2 from amino acid residue 219 to amino acid residue 294 of human CD38, or extends across the corresponding amino acid residues in homologous CD38 from other species.

In one embodiment, the epitope extends at least across a region having SEQ ID NO:2 from amino acid residue 219 to amino acid residue 285 of human CD38, or extends across the corresponding amino acid residues in homologous CD38 from other species.

In one embodiment, the epitope extends at least across a region having SEQ ID NO:2 from amino acid residue 220 to amino acid residue 300 of human CD38, or extends across the corresponding amino acid residues in homologous CD38 from other species.

In one embodiment, the epitope extends at least across a region having SEQ ID NO:2 from amino acid residue 220 to amino acid residue 296 of human CD38, or extends across the corresponding amino acid residues in homologous CD38 from other species.

In one embodiment, the epitope extends at least across a region having SEQ ID NO:2 from amino acid residue 220 to amino acid residue 294 of human CD38, or extends across the corresponding amino acid residues in homologous CD38 from other species.

In one embodiment, the epitope extends at least across a region having SEQ ID NO:2 from amino acid residue 220 to amino acid residue 285 of human CD38, or extends across the corresponding amino acid residues in homologous CD38 from other species.

In one embodiment, the epitope extends at least across a region having SEQ ID NO:2 from amino acid residue 233 to amino acid residue 300 of human CD38, or extends across the corresponding amino acid residues in homologous CD38 from other species.

In one embodiment, the epitope extends at least across a region having SEQ ID NO:2 from amino acid residue 233 to amino acid residue 296 of human CD38, or extends across the corresponding amino acid residues in homologous CD38 from other species.

In one embodiment, the epitope extends at least across a region having SEQ ID NO:2 from amino acid residue 233 to amino acid residue 294 of human CD38, or extends across the corresponding amino acid residues in homologous CD38 from other species.

In one embodiment, the epitope extends at least across a region having SEQ ID NO:2 from amino acid residue 233 to amino acid residue 285 of human CD38, or extends across the corresponding amino acid residues in homologous CD38 from other species.

In one embodiment, the epitope extends at least across a region having SEQ ID NO:2 from amino acid residue 220 to amino acid residue 241 and amino acid residue 273 to amino acid residue 285, or extends across the corresponding amino acid residues in homologous CD38 from other species.

In one embodiment, the epitope comprises at least a polypeptide having the amino acid sequence of SEQ ID NO:2 or the corresponding cysteine in homologous CD38 from other species.

In one embodiment, the epitope comprises at least a polypeptide having the amino acid sequence of SEQ ID NO: cysteine 275 of human CD38 from 2 or the corresponding cysteine in homologous CD38 from other species.

In one embodiment, the epitope comprises at least a polypeptide having the amino acid sequence of SEQ ID NO: cysteine 254 and cysteine 275 of human CD38 from 2 or the corresponding cysteines in homologous CD38 from other species.

In one embodiment, the epitope comprises a sequence referred to as having SEQ ID NO: cysteine 254 and cysteine 275 of human CD38 from 2 or the 5 (penultimate) C-terminal disulfide loop of the corresponding cysteine in homologous CD38 from other species. Specifically, the 5 th (penultimate) C-terminal disulfide loop of human CD38 comprises a disulfide having the amino acid sequence of SEQ ID NO:2 from amino acid residue 220 to amino acid residue 285 of human CD38, or the corresponding cysteine in homologous CD38 from other species.

In one embodiment, the epitope does not comprise a polypeptide having the sequence of SEQ ID NO: cysteine 287 of human CD38 from 2 or the corresponding cysteine in homologous CD38 from other species.

In one embodiment, the epitope does not comprise a polypeptide having the sequence of SEQ ID NO:2, cysteine 290 of human CD38 or the corresponding cysteine in homologous CD38 from other species.

In one embodiment, the epitope does not comprise a polypeptide having the sequence of SEQ ID NO: cysteine 287 and cysteine 290 of human CD38 of 2 or the corresponding cysteines in homologous CD38 from other species.

In one embodiment, the epitope does not comprise a sequence that relates to a polypeptide having the amino acid sequence of SEQ ID NO: cysteine 287 and cysteine 296 of human CD38 from 2 or the 6 th C-terminal disulfide loop of the corresponding cysteine in homologous CD38 from other species. Specifically, the 6 th C-terminal disulfide loop of human CD38 comprises a disulfide having the amino acid sequence of SEQ ID NO:2 from amino acid residue 285 to amino acid residue 300 of human CD38, or the corresponding cysteine in homologous CD38 from other species.

In one embodiment, the compounds of the invention directly protect dopaminergic neurons from the neurotoxin MPP⁺Induced mitochondrial inhibition.

In one embodiment, the compounds of the present invention have at least one, preferably at least two, more preferably three of the following properties:

it protects dopaminergic neurons from energy deficiency (energy deficiency) due to inhibition of mitochondrial complex I, in particular mitochondrial neurotoxin MPP⁺；

It uses human Peripheral Blood Mononuclear Cells (PBMCs) to increase the in vitro release of the anti-inflammatory cytokine interleukin-10 (IL-10); and/or

It increases the level of IL-10 in vivo when injected into mice.

In one embodiment, the compounds of the present invention compete with CD31, preferably human CD31, and induce tyrosine phosphorylation. In particular, the compounds of the present invention induce tyrosine phosphorylation of discrete cytoplasmic matrices that are inhibited in the presence of genistein.

Examples of phosphorylated discrete cytoplasmic matrices include, but are not limited to, the C-cbl protooncogene, the protein kinase syk, the p85 subunit of phosphatidylinositol-3 kinase, phospholipase C-gamma (PLC-. gamma.), Raf-1/MAP kinase, CD 3-zeta/ZAP-70 signaling pathway.

In one embodiment, the compounds of the invention compete with CD31, preferably with human CD31, and trigger CD38 internalization.

In one embodiment, the compounds of the invention compete with CD31, preferably human CD31, and induce lysosomal exocytosis. In particular, the compounds of the present invention induce lysosomal exocytosis, which is inhibited in the presence of vacuolin-1.

In one embodiment, the compounds of the invention do not mediate, trigger or enhance Antibody Dependent Cellular Cytotoxicity (ADCC), Antibody Dependent Cellular Phagocytosis (ADCP) and/or Complement Dependent Cytotoxicity (CDC).

In one embodiment, the compound of the invention is a CD31 peptide, preferably a human CD31 peptide.

In one embodiment, the compound of the invention is a peptide comprising or consisting of the extracellular domain of human CD 31. In one embodiment, the compounds of the invention are fragments of the extracellular domain of human CD 31. In one embodiment, the compounds of the present invention are peptides comprising or consisting of Ig-like domains 1-3 of human CD 31.

In one embodiment, the compound of the invention is a peptide comprising SEQ ID NO:1 or a peptide consisting thereof or a variant thereof. In one embodiment, the compound of the invention is a peptide comprising SEQ ID NO:1 or a peptide consisting thereof or a variant thereof. In one embodiment, the compound of the invention is a peptide comprising SEQ ID NO:1 or a peptide consisting thereof or a variant thereof. In one embodiment, the compound of the invention is a peptide comprising SEQ ID NO:1 or a peptide consisting thereof or a variant thereof. In one embodiment, the compound of the invention is a peptide comprising SEQ ID NO:1 or a peptide consisting thereof or a variant thereof. In one embodiment, the compound of the invention is a peptide comprising SEQ ID NO:1 or a peptide consisting thereof or a variant thereof. In one embodiment, the compound of the invention is a peptide comprising SEQ ID NO:1 or a peptide consisting thereof or a variant thereof. In one embodiment, the compound of the invention is a peptide comprising SEQ ID NO:1 or a peptide consisting thereof or a variant thereof. In one embodiment, the compound of the invention is a peptide comprising SEQ ID NO:1 or a peptide consisting thereof or a variant thereof. In one embodiment, the compound of the invention is a peptide comprising SEQ ID NO:1 or a peptide consisting thereof or a variant thereof. In one embodiment, the compound of the invention is a peptide comprising SEQ ID NO:1 or a peptide consisting thereof or a variant thereof. In one embodiment, the compound of the invention is a peptide comprising SEQ ID NO:1 or a peptide consisting thereof or a variant thereof. In one embodiment, the compound of the invention is a peptide comprising SEQ ID NO:1 or a peptide consisting thereof or a variant thereof. In one embodiment, the compound of the invention is a peptide comprising SEQ ID NO:1 or a peptide consisting thereof or a variant thereof.

In one embodiment, a variant of a peptide as described above shares at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more local identity (local identity) with said peptide.

In one embodiment, the compounds of the invention are chimeric CD31 peptides, preferably chimeric human CD31 peptides.

In one embodiment, the chimeric CD31 peptide comprises or consists of a CD31 peptide as defined above, fused to the Fc domain of an immunoglobulin to form an Fc-fusion protein.

In one embodiment, the chimeric CD31 peptide comprises or consists of a CD31 peptide as defined above, fused to human serum albumin to form a human serum albumin fusion protein.

In one embodiment, the chimeric CD31 peptide comprises a CD31 peptide as defined above fused to one or more domains of human serum albumin to form a human serum albumin domain fusion protein. In particular, the chimeric CD31 peptide may comprise or consist of a CD31 peptide as defined above, fused to domain III of human serum albumin.

As used herein, the term "domain III of human serum albumin" comprises the amino acid sequence as set forth in SEQ ID NO: 43, corresponding to human serum albumin encoded by the ALB gene (Uniprot accession and version: P02768-1, last modified at 4/1 of 1990; Checksum: F88FF61DD242E 818).

In one embodiment, the chimeric CD31 peptide comprises or consists of a CD31 peptide as defined above, fused to transferrin to form a transferrin-fusion protein.

In one embodiment, the compound of the invention is an anti-CD 38 antibody or antigen-binding fragment thereof.

In one embodiment, the compound of the invention is an anti-CD 38 polyclonal antibody or antigen binding fragment thereof.

In one embodiment, the compound of the invention is an anti-CD 38 monoclonal antibody or antigen-binding fragment thereof.

In one embodiment, the anti-CD 38 antibody or antigen-binding fragment thereof of the invention is selected from the group comprising or consisting of: mouse anti-human CD38 monoclonal antibody clone HB 7; rat anti-human CD38 monoclonal antibody clone 90; mouse anti-human CD38 monoclonal antibody clone AT 1; mouse anti-human CD38 monoclonal antibody clone T16; mouse anti-human CD38 monoclonal antibody clone IB 4; and the above mutated, recombinant (including chimeric and humanized), bispecific and modified antibodies capable of specifically competing with CD 31.

In one embodiment, the anti-CD 38 antibody or antigen-binding fragment thereof of the invention is selected from the group comprising or consisting of: mouse anti-human CD38 monoclonal antibody clone HB 7; rat anti-human CD38 monoclonal antibody clone 90; and the above mutated, recombinant (including chimeric and humanized), bispecific and modified antibodies capable of specifically competing with CD 31.

In one embodiment, the anti-CD 38 antibody or antigen-binding fragment thereof of the invention is selected from the group comprising or consisting of: mouse anti-human CD38 monoclonal antibody clone HB 7; and the above mutated, recombinant (including chimeric and humanized), bispecific and modified antibodies capable of specifically competing with CD 31.

Hereinafter, and unless explicitly stated otherwise, CDR numbering and definition are according to Chothia definitions.

In one embodiment, the anti-CD 38 antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region (abbreviated herein as HCVR or V)_H) Comprising at least one, preferably at least two, more preferably three of the following Complementarity Determining Regions (CDRs):

V_H-CDR1：GFTFSNX₁(SEQ ID NO:4) or a variant thereof;

V_H-CDR2：X₂GSSRX₃(SEQ ID NO:5) or a variant thereof; and

V_H-CDR3：X₄X₅X₆X₇X₈YX₉X₁₀X₁₁X₁₂GMDV (SEQ ID NO:6) or a variant thereof,

wherein:

X₁selected from Tyr (Y), Asn (N), and Ser (S);

X₂selected from Ser (S) and Tyr (Y);

X₃selected from Tyr (Y), Asp (D), Asn (N) and Ser (S);

X₄selected from ser(s) and a vacancy;

X₅selected from ser(s) and a vacancy;

X₆selected from Ser (S) and Tyr (Y);

X₇selected from Ser (S) and Asp (D);

X₈selected from Tyr (Y), Ser (S), Asp (D) and Gly (G);

X₉selected from Tyr (Y) and Gly (G);

X₁₀selected from Ser (S), Tyr (Y) and Phe (F);

X₁₁selected from Gly (G) and Asp (D); and is

X₁₂Selected from Asn (N), Tyr (Y) and Ser (S).

In one embodiment, the anti-CD 38 antibody or antigen-binding fragment thereof of the invention comprises a HCVR comprising the following three CDRs:

V_H-CDR1：GFTFSNX₁(SEQ ID NO:4) or a variant thereof;

V_H-CDR2：X₂GSSRX₃(SEQ ID NO:5) or a variant thereof; and

V_H-CDR3：X₄X₅X₆X₇X₈YX₉X₁₀X₁₁X₁₂GMDV (SEQ ID NO:6) or a variant thereof,

wherein:

X₁selected from Tyr (Y), Asn (N), and Ser (S);

X₂selected from Ser (S) and Tyr (Y);

X₃selected from Tyr (Y)Asp (D), Asn (N) and Ser (S);

X₄selected from ser(s) and a vacancy;

X₅selected from ser(s) and a vacancy;

X₆selected from Ser (S) and Tyr (Y);

X₇selected from Ser (S) and Asp (D);

X₈selected from Tyr (Y), Ser (S), Asp (D) and Gly (G);

X₉selected from Tyr (Y) and Gly (G);

X₁₀selected from Ser (S), Tyr (Y) and Phe (F);

X₁₁selected from Gly (G) and Asp (D); and is

X₁₂Selected from Asn (N), Tyr (Y) and Ser (S).

In one embodiment, the anti-CD 38 antibody or antigen-binding fragment thereof according to the invention is designated "clone B08" and comprises a HCVR comprising at least one, preferably at least two, more preferably three of the following CDRs:

V_H-CDR 1: GFTFSNY (SEQ ID NO:7) or a variant thereof;

V_H-CDR 2: SGSSRS (SEQ ID NO:10) or a variant thereof; and

V_H-CDR 3: SSDDYYYDYGMDV (SEQ ID NO:15) or a variant thereof.

In one embodiment, clone B08 comprises a HCVR comprising the following three CDRs:

V_H-CDR 1: GFTFSNY (SEQ ID NO:7) or a variant thereof;

V_H-CDR 2: SGSSRS (SEQ ID NO:10) or a variant thereof; and

V_H-CDR 3: SSDDYYYDYGMDV (SEQ ID NO:15) or a variant thereof.

In one embodiment, the anti-CD 38 antibody or antigen-binding fragment thereof according to the invention is designated "clone C05" and comprises a HCVR comprising at least one, preferably at least two, more preferably three of the following CDRs:

V_H-CDR 1: GFTFSNN (SEQ ID NO:8) or a variant thereof;

V_H-CDR 2: SGSSRY (SEQ ID NO:11) or a variant thereof; and

V_H-CDR 3: SSSSYYYSGNGMDV (SEQ ID NO:16) or a variant thereof.

In one embodiment, clone C05 comprises a HCVR comprising the following three CDRs:

V_H-CDR 1: GFTFSNN (SEQ ID NO:8) or a variant thereof;

V_H-CDR 2: SGSSRY (SEQ ID NO:11) or a variant thereof; and

V_H-CDR 3: SSSSYYYSGNGMDV (SEQ ID NO:16) or a variant thereof.

In one embodiment, the anti-CD 38 antibody or antigen-binding fragment thereof according to the invention is designated "clone C06" and comprises a HCVR comprising at least one, preferably at least two, more preferably three of the following CDRs:

V_H-CDR 1: GFTFSNS (SEQ ID NO:9) or a variant thereof;

V_H-CDR 2: SGSSRN (SEQ ID NO:12) or variants thereof; and

V_H-CDR 3: SSYSSYGSGNGMDV (SEQ ID NO:17) or a variant thereof.

In one embodiment, clone C06 comprises a HCVR comprising the following three CDRs:

V_H-CDR 1: GFTFSNS (SEQ ID NO:9) or a variant thereof;

V_H-CDR 2: SGSSRN (SEQ ID NO:12) or variants thereof; and

V_H-CDR 3: SSYSSYGSGNGMDV (SEQ ID NO:17) or a variant thereof.

In one embodiment, the anti-CD 38 antibody or antigen-binding fragment thereof according to the invention is designated "clone D06" and comprises a HCVR comprising at least one, preferably at least two, more preferably three of the following CDRs:

V_H-CDR 1: GFTFSNY (SEQ ID NO:7) or a variant thereof;

V_H-CDR 2: YGSSRD (SEQ ID NO:13) or variants thereof; and

V_H-CDR3：SSGYYFGYGMDV(SEQ ID NO：19) or a variant thereof.

In one embodiment, clone D06 comprises a HCVR comprising the following three CDRs:

V_H-CDR 1: GFTFSNY (SEQ ID NO:7) or a variant thereof;

V_H-CDR 2: YGSSRD (SEQ ID NO:13) or variants thereof; and

V_H-CDR 3: SSGYYFGYGMDV (SEQ ID NO:19) or a variant thereof.

In one embodiment, the anti-CD 38 antibody or antigen-binding fragment thereof according to the invention is designated "clone D07" and comprises a HCVR comprising at least one, preferably at least two, more preferably three of the following CDRs:

V_H-CDR 1: GFTFSNS (SEQ ID NO:9) or a variant thereof;

V_H-CDR 2: SGSSRD (SEQ ID NO:14) or a variant thereof; and

V_H-CDR 3: SSSSYYYSGSGMDV (SEQ ID NO:20) or a variant thereof.

In one embodiment, clone D07 comprises a HCVR comprising the following three CDRs:

V_H-CDR 1: GFTFSNS (SEQ ID NO:9) or a variant thereof;

V_H-CDR 2: SGSSRD (SEQ ID NO:14) or a variant thereof; and

V_H-CDR 3: SSSSYYYSGSGMDV (SEQ ID NO:20) or a variant thereof.

In one embodiment, the anti-CD 38 antibody or antigen-binding fragment thereof according to the invention is designated "clone D10" and comprises a HCVR comprising at least one, preferably at least two, more preferably three of the following CDRs:

V_H-CDR 1: GFTFSNS (SEQ ID NO:9) or a variant thereof;

V_H-CDR 2: SGSSRD (SEQ ID NO:14) or a variant thereof; and

V_H-CDR 3: SSSSYYYSGNGMDV (SEQ ID NO:16) or a variant thereof.

In one embodiment, clone D10 comprises a HCVR comprising the following three CDRs:

V_H-CDR 1: GFTFSNS (SEQ ID NO:9) or a variant thereof;

V_H-CDR 2: SGSSRD (SEQ ID NO:14) or a variant thereof; and

V_H-CDR 3: SSSSYYYSGNGMDV (SEQ ID NO:16) or a variant thereof.

In one embodiment, the anti-CD 38 antibody or antigen-binding fragment thereof of the invention comprises a light chain variable region (abbreviated herein as LCVR or V)_L) Comprising at least one, preferably at least two, more preferably three of the following Complementarity Determining Regions (CDRs):

V_L-CDR1：AGTSSDVGGX₁₃X₁₄X₁₅VS (SEQ ID NO:21) or a variant thereof;

V_L-CDR2：X₁₆DSX₁₇RPS (SEQ ID NO:22) or a variant thereof; and

V_L-CDR 3: STRVFGGGT (SEQ ID NO:23) or a variant thereof,

wherein:

X₁₃selected from Ser (S) and Asn (N);

X₁₄selected from Ser (S) and Tyr (Y);

X₁₅selected from Tyr (Y) and Ser (S);

X₁₆selected from Tyr (Y), Ser (S), and Asp (D); and is

X₁₇Selected from Tyr (Y) and Asn (N).

In one embodiment, the anti-CD 38 antibody or antigen-binding fragment thereof of the invention comprises a LCVR comprising the following three CDRs:

V_L-CDR1：AGTSSDVGGX₁₃X₁₄X₁₅VS (SEQ ID NO:21) or a variant thereof;

V_L-CDR2：X₁₆DSX₁₇RPS (SEQ ID NO:22) or a variant thereof; and

V_L-CDR 3: STRVFGGGT (SEQ ID NO:23) or a variant thereof,

wherein:

X₁₃selected from Ser (S) and Asn (N);

X₁₄selected from Ser (S) and Tyr (Y);

X₁₅selected from Tyr (Y) and Ser (S);

X₁₆selected from Tyr (Y), Ser (S), and Asp (D); and is

X₁₇Selected from Tyr (Y) and Asn (N).

In one embodiment, the anti-CD 38 antibody or antigen-binding fragment thereof according to the invention is designated "clone B08" and comprises an LCVR comprising at least one, preferably at least two, more preferably three of the following CDRs:

V_L-CDR 1: AGTSSDVGGSYSVS (SEQ ID NO:24) or a variant thereof;

V_L-CDR 2: DDSNRPS (SEQ ID NO:29) or a variant thereof; and

V_L-CDR 3: STRVFGGGT (SEQ ID NO:23) or a variant thereof.

In one embodiment, clone B08 comprises an LCVR comprising the following three CDRs:

V_L-CDR 1: AGTSSDVGGSYSVS (SEQ ID NO:24) or a variant thereof;

V_L-CDR 2: DDSNRPS (SEQ ID NO:29) or a variant thereof; and

V_L-CDR 3: STRVFGGGT (SEQ ID NO:23) or a variant thereof.

In one embodiment, the anti-CD 38 antibody or antigen-binding fragment thereof according to the invention is designated "clone C05" and comprises an LCVR comprising at least one, preferably at least two, more preferably three of the following CDRs:

V_L-CDR 1: AGTSSDVGGSSYVS (SEQ ID NO:25) or a variant thereof;

V_L-CDR 2: YDSYRPS (SEQ ID NO:30) or variants thereof; and

V_L-CDR 3: STRVFGGGT (SEQ ID NO:23) or a variant thereof.

In one embodiment, clone C05 comprises an LCVR comprising the following three CDRs:

V_L-CDR 1: AGTSSDVGGSSYVS (SEQ ID NO:25) or a variant thereof;

V_L-CDR 2: YDSYRPS (SEQ ID NO:30) or variants thereof; and

V_L-CDR 3: STRVFGGGT (SEQ ID NO:23) or a variant thereof.

In one embodiment, the anti-CD 38 antibody or antigen-binding fragment thereof according to the invention is designated "clone C06" and comprises an LCVR comprising at least one, preferably at least two, more preferably three of the following CDRs:

V_L-CDR 1: AGTSSDVGGSSYVS (SEQ ID NO:25) or a variant thereof;

V_L-CDR 2: YDSYRPS (SEQ ID NO:30) or variants thereof; and

V_L-CDR 3: STRVFGGGT (SEQ ID NO:23) or a variant thereof.

In one embodiment, clone C06 comprises an LCVR comprising the following three CDRs:

V_L-CDR 1: AGTSSDVGGSSYVS (SEQ ID NO:25) or a variant thereof;

V_L-CDR 2: YDSYRPS (SEQ ID NO:30) or variants thereof; and

V_L-CDR 3: STRVFGGGT (SEQ ID NO:23) or a variant thereof.

In one embodiment, the anti-CD 38 antibody or antigen-binding fragment thereof according to the invention is designated "clone D06" and comprises an LCVR comprising at least one, preferably at least two, more preferably three of the following CDRs:

V_L-CDR 1: AGTSSDVGGSYYVS (SEQ ID NO:27) or a variant thereof;

V_L-CDR 2: SDSYRPS (SEQ ID NO:32) or variants thereof; and

V_L-CDR 3: STRVFGGGT (SEQ ID NO:23) or a variant thereof.

In one embodiment, clone D06 comprises an LCVR comprising the following three CDRs:

V_L-CDR 1: AGTSSDVGGSYYVS (SEQ ID NO:27) or a variant thereof;

V_L-CDR 2: SDSYRPS (SEQ ID NO:32) or variants thereof; and

V_L-CDR 3: STRVFGGGT (SEQ ID NO:23) or a variant thereof.

In one embodiment, the anti-CD 38 antibody or antigen-binding fragment thereof according to the invention is designated "clone D07" and comprises an LCVR comprising at least one, preferably at least two, more preferably three of the following CDRs:

V_L-CDR 1: AGTSSDVGGNSYVS (SEQ ID NO:28) or a variant thereof;

V_L-CDR 2: YDSYRPS (SEQ ID NO:30) or variants thereof; and

V_L-CDR 3: STRVFGGGT (SEQ ID NO:23) or a variant thereof.

In one embodiment, clone D07 comprises an LCVR comprising the following three CDRs:

V_L-CDR 1: AGTSSDVGGNSYVS (SEQ ID NO:28) or a variant thereof;

V_L-CDR 2: YDSYRPS (SEQ ID NO:30) or variants thereof; and

V_L-CDR 3: STRVFGGGT (SEQ ID NO:23) or a variant thereof.

In one embodiment, the anti-CD 38 antibody or antigen-binding fragment thereof according to the invention is designated "clone D10" and comprises an LCVR comprising at least one, preferably at least two, more preferably three of the following CDRs:

V_L-CDR 1: AGTSSDVGGNSYVS (SEQ ID NO:28) or a variant thereof;

V_L-CDR 2: YDSYRPS (SEQ ID NO:30) or variants thereof; and

V_L-CDR 3: STRVFGGGT (SEQ ID NO:23) or a variant thereof.

In one embodiment, clone D10 comprises an LCVR comprising the following three CDRs:

V_L-CDR 1: AGTSSDVGGNSYVS (SEQ ID NO:28) or a variant thereof;

V_L-CDR 2: YDSYRPS (SEQ ID NO:30) or variants thereof; and

V_L-CDR 3: STRVFGGGT (SEQ ID NO:23) or a variant thereof.

In one embodiment, the anti-CD 38 antibody or antigen-binding fragment thereof of the invention comprises:

-a HCVR comprising at least one, preferably at least two, more preferably three of the following CDRs:

V_H-CDR1：GFTFSNX₁(SEQ ID NO:4) or a variant thereof,

V_H-CDR2：X₂GSSRX₃(SEQ ID NO:5) or a variant thereof, and

V_H-CDR3：X₄X₅X₆X₇X₈YX₉X₁₀X₁₁X₁₂GMDV (SEQ ID NO:6) or a variant thereof; and

-an LCVR comprising at least one, preferably at least two, more preferably three, of the following CDRs:

V_L-CDR1：AGTSSDVGGX₁₃X₁₄X₁₅VS (SEQ ID NO:21) or a variant thereof;

V_L-CDR2：X₁₆DSX₁₇RPS (SEQ ID NO:22) or a variant thereof; and

V_L-CDR 3: STRVFGGGT (SEQ ID NO:23) or a variant thereof;

wherein:

X₁selected from Tyr (Y), Asn (N), and Ser (S);

X₂selected from Ser (S) and Tyr (Y);

X₃selected from Tyr (Y), Asp (D), Asn (N) and Ser (S);

X₄selected from ser(s) and a vacancy;

X₅selected from ser(s) and a vacancy;

X₆selected from Ser (S) and Tyr (Y);

X₇selected from Ser (S) and Asp (D);

X₈selected from Tyr (Y), Ser (S), Asp (D) and Gly (G);

X₉selected from Tyr (Y) and Gly (G);

X₁₀selected from Ser (S), Tyr (Y) and Phe (F);

X₁₁selected from Gly (G) and Asp (D);

X₁₂selected from Asn (N), Tyr (Y) and Ser (S);

X₁₃selected from Ser (S) and Asn (N);

X₁₄is selected fromSer (S) and Tyr (Y);

X₁₅selected from Tyr (Y) and Ser (S);

X₁₆selected from Tyr (Y), Ser (S), and Asp (D); and is

X₁₇Selected from Tyr (Y) and Asn (N).

In one embodiment, the anti-CD 38 antibody or antigen-binding fragment thereof of the invention comprises:

-HCVR comprising the following three CDRs:

V_H-CDR1：GFTFSNX₁(SEQ ID NO:4) or a variant thereof,

V_H-CDR2：X₂GSSRX₃(SEQ ID NO:5) or a variant thereof, and

V_H-CDR3：X₄X₅X₆X₇X₈YX₉X₁₀X₁₁X₁₂GMDV (SEQ ID NO:6) or a variant thereof; and

-an LCVR comprising the following three CDRs:

V_L-CDR1：AGTSSDVGGX₁₃X₁₄X₁₅VS (SEQ ID NO:21) or a variant thereof;

V_L-CDR2：X₁₆DSX₁₇RPS (SEQ ID NO:22) or a variant thereof; and

V_L-CDR 3: STRVFGGGT (SEQ ID NO:23) or a variant thereof;

wherein:

X₁selected from Tyr (Y), Asn (N), and Ser (S);

X₂selected from Ser (S) and Tyr (Y);

X₃selected from Tyr (Y), Asp (D), Asn (N) and Ser (S);

X₄selected from ser(s) and a vacancy;

X₅selected from ser(s) and a vacancy;

X₆selected from Ser (S) and Tyr (Y);

X₇selected from Ser (S) and Asp (D);

X₈selected from Tyr (Y), Ser (S), Asp (D) and Gly (G);

X₉selected from Tyr (Y) and Gly: (G)；

X₁₀Selected from Ser (S), Tyr (Y) and Phe (F);

X₁₁selected from Gly (G) and Asp (D);

X₁₂selected from Asn (N), Tyr (Y) and Ser (S);

X₁₃selected from Ser (S) and Asn (N);

X₁₄selected from Ser (S) and Tyr (Y);

X₁₅selected from Tyr (Y) and Ser (S);

X₁₆selected from Tyr (Y), Ser (S), and Asp (D); and is

X₁₇Selected from Tyr (Y) and Asn (N).

In one embodiment, the anti-CD 38 antibody or antigen-binding fragment thereof of the invention is designated "clone B08" and comprises:

-a HCVR comprising at least one, preferably at least two, more preferably three of the following CDRs:

V_H-CDR 1: GFTFSNY (SEQ ID NO:7) or a variant thereof,

V_H-CDR 2: SGSSRS (SEQ ID NO:10) or variant thereof, and

V_H-CDR 3: SSDDYYYDYGMDV (SEQ ID NO:15) or a variant thereof; and

-an LCVR comprising at least one, preferably at least two, more preferably three, of the following CDRs:

V_L-CDR 1: AGTSSDVGGSYSVS (SEQ ID NO:24) or variants thereof,

V_L-CDR 2: DDSNRPS (SEQ ID NO:29) or a variant thereof, and

V_L-CDR 3: STRVFGGGT (SEQ ID NO:23) or a variant thereof.

In one embodiment, clone B08 comprises:

-HCVR comprising the following three CDRs:

V_H-CDR 1: GFTFSNY (SEQ ID NO:7) or a variant thereof,

V_H-CDR 2: SGSSRS (SEQ ID NO:10) or variant thereof, and

V_H-CDR 3: SSDDYYYDYGMDV (SEQ ID NO:15) or a salt thereofA variant; and

-an LCVR comprising the following three CDRs:

V_L-CDR 1: AGTSSDVGGSYSVS (SEQ ID NO:24) or variants thereof,

V_L-CDR 2: DDSNRPS (SEQ ID NO:29) or a variant thereof, and

V_L-CDR 3: STRVFGGGT (SEQ ID NO:23) or a variant thereof.

In one embodiment, the anti-CD 38 antibody or antigen-binding fragment thereof of the invention is named "clone C05" and comprises:

-a HCVR comprising at least one, preferably at least two, more preferably three of the following CDRs:

V_H-CDR 1: GFTFSNN (SEQ ID NO:8) or a variant thereof,

V_H-CDR 2: SGSSRY (SEQ ID NO:11) or variants thereof, and

V_H-CDR 3: SSSSYYYSGNGMDV (SEQ ID NO:16) or a variant thereof; and

-an LCVR comprising at least one, preferably at least two, more preferably three, of the following CDRs:

V_L-CDR 1: AGTSSDVGGSSYVS (SEQ ID NO:25) or a variant thereof,

V_L-CDR 2: YDSYRPS (SEQ ID NO:30) or variants thereof, and

V_L-CDR 3: STRVFGGGT (SEQ ID NO:23) or a variant thereof.

In one embodiment, clone C05 comprises:

-HCVR comprising the following three CDRs:

V_H-CDR 1: GFTFSNN (SEQ ID NO:8) or a variant thereof,

V_H-CDR 2: SGSSRY (SEQ ID NO:11) or variants thereof, and

V_H-CDR 3: SSSSYYYSGNGMDV (SEQ ID NO:16) or a variant thereof; and

-an LCVR comprising the following three CDRs:

V_L-CDR 1: AGTSSDVGGSSYVS (SEQ ID NO:25) or a variant thereof,

V_L-CDR2：YDSYRPS(SEQ ID NO:30) or a variant thereof, and

V_L-CDR 3: STRVFGGGT (SEQ ID NO:23) or a variant thereof.

In one embodiment, the anti-CD 38 antibody or antigen-binding fragment thereof of the invention is named "clone C06" and comprises:

-a HCVR comprising at least one, preferably at least two, more preferably three of the following CDRs:

V_H-CDR 1: GFTFSNS (SEQ ID NO:9) or a variant thereof,

V_H-CDR 2: SGSSRN (SEQ ID NO:12) or variants thereof, and

V_H-CDR 3: SSYSSYGSGNGMDV (SEQ ID NO:17) or a variant thereof; and

-an LCVR comprising at least one, preferably at least two, more preferably three, of the following CDRs:

V_L-CDR 1: AGTSSDVGGSSYVS (SEQ ID NO:25) or a variant thereof,

V_L-CDR 2: YDSYRPS (SEQ ID NO:30) or variants thereof, and

V_L-CDR 3: STRVFGGGT (SEQ ID NO:23) or a variant thereof.

In one embodiment, clone C06 comprises:

-HCVR comprising the following three CDRs:

V_H-CDR 1: GFTFSNS (SEQ ID NO:9) or a variant thereof,

V_H-CDR 2: SGSSRN (SEQ ID NO:12) or variants thereof, and

V_H-CDR 3: SSYSSYGSGNGMDV (SEQ ID NO:17) or a variant thereof; and

-an LCVR comprising the following three CDRs:

V_L-CDR 1: AGTSSDVGGSSYVS (SEQ ID NO:25) or a variant thereof,

V_L-CDR 2: YDSYRPS (SEQ ID NO:30) or variants thereof, and

V_L-CDR 3: STRVFGGGT (SEQ ID NO:23) or a variant thereof.

In one embodiment, the anti-CD 38 antibody or antigen-binding fragment thereof according to the invention is designated "clone D06" and comprises:

-a HCVR comprising at least one, preferably at least two, more preferably three of the following CDRs:

V_H-CDR 1: GFTFSNY (SEQ ID NO:7) or a variant thereof,

V_H-CDR 2: YGSSRD (SEQ ID NO:13) or variants thereof, and

V_H-CDR 3: SSGYYFGYGMDV (SEQ ID NO:19) or a variant thereof; and

-an LCVR comprising at least one, preferably at least two, more preferably three, of the following CDRs:

V_L-CDR 1: AGTSSDVGGSYYVS (SEQ ID NO:27) or a variant thereof,

V_L-CDR 2: SDSYRPS (SEQ ID NO:32) or variants thereof, and

V_L-CDR 3: STRVFGGGT (SEQ ID NO:23) or a variant thereof.

In one embodiment, clone D06 comprises:

-HCVR comprising the following three CDRs:

V_H-CDR 1: GFTFSNY (SEQ ID NO:7) or a variant thereof,

V_H-CDR 2: YGSSRD (SEQ ID NO:13) or variants thereof, and

V_H-CDR 3: SSGYYFGYGMDV (SEQ ID NO:19) or a variant thereof; and

-an LCVR comprising the following three CDRs:

V_L-CDR 1: AGTSSDVGGSYYVS (SEQ ID NO:27) or a variant thereof,

V_L-CDR 2: SDSYRPS (SEQ ID NO:32) or variants thereof, and

V_L-CDR 3: STRVFGGGT (SEQ ID NO:23) or a variant thereof.

In one embodiment, the anti-CD 38 antibody or antigen-binding fragment thereof according to the invention is designated "clone D07" and comprises:

-a HCVR comprising at least one, preferably at least two, more preferably three of the following CDRs:

V_H-CDR 1: GFTFSNS (SEQ ID NO:9) or variants thereof，

V_H-CDR 2: SGSSRD (SEQ ID NO:14) or a variant thereof, and

V_H-CDR 3: SSSSYYYSGSGMDV (SEQ ID NO:20) or a variant thereof; and

-an LCVR comprising at least one, preferably at least two, more preferably three, of the following CDRs:

V_L-CDR 1: AGTSSDVGGNSYVS (SEQ ID NO:28) or variants thereof,

V_L-CDR 2: YDSYRPS (SEQ ID NO:30) or variants thereof, and

V_L-CDR 3: STRVFGGGT (SEQ ID NO:23) or a variant thereof.

In one embodiment, clone D07 comprises:

-HCVR comprising the following three CDRs:

V_H-CDR 1: GFTFSNS (SEQ ID NO:9) or a variant thereof,

V_H-CDR 2: SGSSRD (SEQ ID NO:14) or a variant thereof, and

V_H-CDR 3: SSSSYYYSGSGMDV (SEQ ID NO:20) or a variant thereof; and

-an LCVR comprising the following three CDRs:

V_L-CDR 1: AGTSSDVGGNSYVS (SEQ ID NO:28) or variants thereof,

V_L-CDR 2: YDSYRPS (SEQ ID NO:30) or variants thereof, and

V_L-CDR 3: STRVFGGGT (SEQ ID NO:23) or a variant thereof.

In one embodiment, the anti-CD 38 antibody or antigen-binding fragment thereof according to the invention is designated "clone D10" and comprises:

-a HCVR comprising at least one, preferably at least two, more preferably three of the following CDRs:

V_H-CDR 1: GFTFSNS (SEQ ID NO:9) or a variant thereof,

V_H-CDR 2: SGSSRD (SEQ ID NO:14) or a variant thereof, and

V_H-CDR 3: SSSSYYYSGNGMDV (SEQ ID NO:16) or a variant thereof; and

-an LCVR comprising at least one, preferably at least two, more preferably three, of the following CDRs:

V_L-CDR 1: AGTSSDVGGNSYVS (SEQ ID NO:28) or variants thereof,

V_L-CDR 2: YDSYRPS (SEQ ID NO:30) or variants thereof, and

V_L-CDR 3: STRVFGGGT (SEQ ID NO:23) or a variant thereof.

In one embodiment, clone D10 comprises:

-HCVR comprising the following three CDRs:

V_H-CDR 1: GFTFSNS (SEQ ID NO:9) or a variant thereof,

V_H-CDR 2: SGSSRD (SEQ ID NO:14) or a variant thereof, and

V_H-CDR 3: SSSSYYYSGNGMDV (SEQ ID NO:16) or a variant thereof; and

-an LCVR comprising the following three CDRs:

V_L-CDR 1: AGTSSDVGGNSYVS (SEQ ID NO:28) or variants thereof,

V_L-CDR 2: YDSYRPS (SEQ ID NO:30) or variants thereof, and

V_L-CDR 3: STRVFGGGT (SEQ ID NO:23) or a variant thereof.

A variant of a CDR refers to any CDR as defined above, characterized by having 1,2 or 3 amino acids substituted by different amino acids.

A variant of a CDR refers to any CDR as defined above, characterized by having an amino acid sequence that shares at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with a particular CDR.

In one embodiment, the anti-CD 38 antibody or antigen-binding fragment thereof according to the invention is designated "clone B08" and comprises a heavy chain variable region having the sequence set forth in SEQ ID NO:33, or a variant thereof.

In one embodiment, the anti-CD 38 antibody or antigen-binding fragment thereof according to the invention is designated "clone C05" and comprises a heavy chain variable region having the sequence set forth in SEQ ID NO:34, or a variant thereof.

In one embodiment, the anti-CD 38 antibody or antigen-binding fragment thereof described herein is named "clone C06" and comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO:35, or a variant thereof.

In one embodiment, the anti-CD 38 antibody or antigen-binding fragment thereof according to the invention is designated "clone D06" and comprises a HCVR as shown in SEQ ID NO:36 or variant thereof according to the following examples.

In one embodiment, the anti-CD 38 antibody or antigen-binding fragment thereof according to the invention is designated "clone D07" and comprises a heavy chain variable region having the sequence set forth in SEQ ID NO:37, or a variant thereof.

In one embodiment, the anti-CD 38 antibody or antigen-binding fragment thereof according to the invention is designated "clone D10" and comprises a heavy chain variable region having the sequence set forth in SEQ ID NO:38, or a variant thereof.

In one embodiment, the anti-CD 38 antibody or antigen-binding fragment thereof according to the invention is designated "clone C06" and comprises a heavy chain variable region having the sequence set forth in SEQ ID NO:40, or a variant thereof.

In one embodiment, the anti-CD 38 antibody or antigen-binding fragment thereof according to the invention is designated "clone D06" and comprises a heavy chain variable region having the sequence set forth in SEQ ID NO:41, or a variant thereof.

In one embodiment, the anti-CD 38 antibody or antigen-binding fragment thereof of the invention comprises:

-HCVR as defined above; and

-an LCVR as defined above.

In one embodiment, the anti-CD 38 antibody or antigen-binding fragment thereof of the invention comprises:

-an HCVR selected from SEQ ID NOs 33, 34, 35, 36, 37 and 38; and

-an LCVR selected from SEQ ID NO 39, 40, 41 and 42.

In one embodiment, the anti-CD 38 antibody or antigen-binding fragment thereof of the invention is designated "clone B08" and comprises:

-a HCVR having the sequence shown as SEQ ID No. 33; and

LCVR with the sequence shown as SEQ ID NO. 39.

In one embodiment, the anti-CD 38 antibody or antigen-binding fragment thereof of the present invention was named "clone C05" and comprises:

-a HCVR having the sequence shown as SEQ ID No. 34; and

LCVR with the sequence shown in SEQ ID NO 40.

In one embodiment, the anti-CD 38 antibody or antigen-binding fragment thereof of the present invention was named "clone C06" and comprises:

-a HCVR having the sequence shown as SEQ ID No. 35; and

LCVR with the sequence shown in SEQ ID NO 40.

In one embodiment, the anti-CD 38 antibody or antigen-binding fragment thereof according to the invention is designated "clone D06" and comprises:

-a HCVR having the sequence shown as SEQ ID No. 36; and

LCVR with the sequence shown in SEQ ID NO. 41.

In one embodiment, the anti-CD 38 antibody or antigen-binding fragment thereof according to the invention is designated "clone D07" and comprises:

-HCVR having the sequence shown as SEQ ID No. 37; and

LCVR with the sequence shown in SEQ ID NO 42.

In one embodiment, the anti-CD 38 antibody or antigen-binding fragment thereof according to the invention is designated "clone D10" and comprises:

-a HCVR having the sequence shown as SEQ ID No. 38; and

LCVR with the sequence shown in SEQ ID NO 42.

A variant of an HCVR and/or LCVR refers to any HCVR and/or LCVR as defined above, which is characterized by having an amino acid sequence that shares at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with a particular HCVR and/or LCVR.

A variant of an HCVR and/or LCVR refers to any HCVR and/or LCVR as defined above, characterized by having 1,2,3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids substituted by different amino acids.

In one embodiment, the anti-CD 38 antibodies or antigen-binding fragments thereof described herein do not have significant affinity and therefore do not have significant ability to bind to CD 3.In one embodiment, the anti-CD 38 antibody or antigen-binding fragment thereof of the invention is not an anti-CD 3/anti-CD 38 bispecific antibody. Illustratively, anti-CD 3/anti-CD 38 bispecific antibodies are disclosed in WO 2016071355.

In one embodiment, the anti-CD 38 antibodies or antigen-binding fragments thereof described herein are chimeric.

In one embodiment, the chimeric anti-CD 38 antibodies or antigen-binding fragments thereof described herein comprise a heavy and/or light chain comprising human constant regions and variable regions from another species, e.g., murine variable regions.

In one embodiment, the anti-CD 38 antibody or antigen-binding fragment thereof of the invention is humanized.

In one embodiment, the anti-CD 38 antibody or antigen-binding fragment thereof of the invention is humanized, preferably a humanized monoclonal antibody or antigen-binding fragment, more preferably comprising:

-a human kappa Light Chain Constant Region (LCCR) derived LCCR, and

-HCCR derived from human IgG1, IgG2, IgG3 or IgG4 (wild-type or mutated) Heavy Chain Constant Region (HCCR).

-a human kappa Light Chain Constant Region (LCCR) derived LCCR, and

heavy Chain Constant Region (HCCR) derived from human IgGl comprising a D297A or a D297G mutation or from human IgG4 comprising a S228P mutation.

The humanized anti-CD 38 antibodies or antigen-binding fragments thereof of the present invention have the advantage of being less immunogenic than murine antibodies (or completely non-immunogenic) when administered to a human subject.

As is well known to those skilled in the art, the choice of HCCR IgG isotype focuses on whether a specific function is required and whether a suitable in vivo half-life is required. For example, antibodies designed to selectively eradicate cancer cells generally require an active isotype that allows complement activation and kills effector-mediated cells through antibody-dependent cell-mediated cytotoxicity. Both the human IgG1 and IgG3 (shorter half-life) isotypes meet these criteria, particularly the human IgG1 isotype (wild-type and variant). In particular, antibodies can be cytotoxic to cells by CDC, ADCC and/or ADCP mechanisms, with the IgG isotypes of HCCR (especially human wild-type and variant IgG1 isotypes) (Salfeld,2007.Nat Biotechnol.25(12): 1369-72; Irani et al, 2015.Mol Immunol.67(2Pt A): 171-82). In fact, the fragment crystallizable (Fc) region interacts with a variety of accessory molecules to mediate indirect effector functions such as antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC).

In one embodiment, the anti-CD 38 antibodies or antigen-binding fragments thereof described herein may be modified to enhance ADCC, ADCP and/or CDC. Such modifications are well known in the art.

For example, antibodies comprising low fucose content are known to enhance ADCC response through Fc γ RIII receptors (see, e.g., international patent publication WO 2014140322). Thus, the antibodies of the invention may comprise a low fucose content.

The term "fucose content" as used herein refers to the percentage of fucosylated forms within the N-glycans attached to the N297 residue of the Fc fragment of each heavy chain of each antibody.

As used herein, the term "low fucose content" refers to a fucose content of less than or equal to 65%. Advantageously, the fucose content is less than or equal to 65%, preferably less than or equal to 60%, 55% or 50%, or even less than or equal to 45%, 40%, 35%, 30%, 25% or 20%. However, the fucose content need not be zero, and it may be, for example, greater than or equal to 5%, 10%, 15%, or 20%.

In one embodiment, the anti-CD 38 antibodies or antigen-binding fragments thereof described herein may further comprise different types of glycosylation (oligomannose) or biantennary (biantennary) complex type N-glycans with variable ratios of bisecting N-acetylglucosamine (GlcNAc) residues or galactose residues in the case of biantennary complex type N-glycans, provided they have a low fucose content (see, e.g., international patent publication WO 2007048077). For example, antibodies with slightly fucosylated N-glycans can be obtained as described in european patent publication EP1176195 or international patent publication WO2001077181 or WO 2012041768.

Oligomannose type N-glycans have a shorter half-life in vivo than do bis-antennary complex type N-glycans. Thus, advantageously, the anti-CD 38 antibody or antigen-binding fragment thereof according to the invention has a glycan structure of the biantennary complex type at the N-glycosylation site of its Fc fragment, which has a low fucose content as defined above.

In one embodiment, an anti-CD 38 antibody or antigen-binding fragment thereof described herein is modified to facilitate delivery across the Blood Brain Barrier (BBB). Means and methods of modifying antibodies to facilitate their passage across the BBB (e.g., when administered parenterally) are well known in the art and are described, for example, in Yu et al, 2011.Sci trans med.3(84):84ra 44; atwal et al, 2011.Sci Transl Med.3(84):84ra 43; and international patent applications WO2015031673 and WO 2016208695.

In some embodiments, the anti-CD 38 antibodies or antigen-binding fragments thereof described herein are conjugated to a therapeutic moiety, i.e., a drug. In one embodiment, the therapeutic moiety is selected from the group consisting of a cytotoxin, a chemotherapeutic agent, a cytokine, an immunosuppressive agent, an immunostimulatory agent, a lytic peptide, and a radioisotope. Such conjugates are referred to herein as "antibody drug conjugates" or "ADCs".

In one embodiment, the compound of the invention is an isolated nucleic acid encoding an anti-CD 38 antibody or antigen-binding fragment thereof as described above.

In one embodiment, the isolated nucleic acid is purified.

In one embodiment, the isolated nucleic acid is purified to:

(1) nucleic acid weight ratios greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95% or greater as determined by absorbance or fluorescence methods (e.g., by measuring absorbance ratios (A) at 260 and 280nm_260/280) And most preferably greater than 96%, 97%, 98% or 99% by weight; or

(2) Homogenization (homogeneity), as shown by agarose gel electrophoresis and the use of intercalating agents such as ethidium bromide, SYBR Green, GelGreen, etc.

It is readily understood that one skilled in the art can design nucleic acid sequences encoding any of the HCVRs and/or LCVRs disclosed herein.

It will also be appreciated that the skilled person is familiar with molecular biological methods intended to modify nucleic acid sequences to improve, for example, recombination productivity, for example by codon optimisation. Finally, the present application encompasses any nucleic acid encoding any HCVR and/or LCVR disclosed herein.

In one embodiment, the compound of the invention is an expression vector comprising an isolated nucleic acid encoding the anti-CD 38 antibody or antigen-binding fragment thereof described above.

In one embodiment, the expression vector of the invention comprises a sequence encoding a HCVR of an antibody or binding fragment thereof of the invention operably linked to a regulatory element.

In one embodiment, the expression vector of the invention comprises a sequence encoding a LCVR of an antibody or binding fragment thereof of the invention operably linked to regulatory elements.

In one embodiment, the expression vector of the present invention comprises:

-a sequence encoding a HCVR of an antibody or binding fragment thereof of the invention, operably linked to a regulatory element, and

-a sequence encoding a LCVR of an antibody or binding fragment thereof according to the invention, operably linked to regulatory elements.

In one embodiment, the compounds of the invention are antigen binding antibody mimetics.

All embodiments of the antibodies or antigen-binding fragments thereof disclosed herein are converted, mutatis mutandis, to antigen-binding antibody mimetics as described in the present invention.

In one embodiment, the compounds of the invention are small organic molecules.

The present invention also relates to compositions comprising, consisting essentially of, or consisting of a compound of the present invention that specifically competes for binding to CD38 with CD 31.

The invention also relates to pharmaceutical compositions comprising compounds of the invention that specifically compete with CD31 for binding to CD 38; and at least one pharmaceutically acceptable excipient.

The term "pharmaceutically acceptable excipient" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The excipient does not produce adverse reactions, allergic reactions or other adverse reactions when administered to an animal, preferably a human. For human administration, the preparations should meet sterility, pyrogenicity, and general safety and purity standards as required by regulatory agencies such as the FDA office or EMA.

Pharmaceutically acceptable excipients that may be used in these pharmaceutical compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids (partial glyceride mixtures), water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances (e.g., sodium carboxymethylcellulose), polyethylene glycol, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.

In one embodiment, the pharmaceutical composition of the invention comprises a pharmaceutically acceptable vehicle for a formulation capable of injection into a subject. These may be, in particular, isotonic, sterile salt solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride, etc. or mixtures of such salts), or dry, in particular freeze-dried, compositions, which, depending on the case, may be supplemented with sterile water or physiological saline to form injectable solutions.

The invention also relates to a medicament comprising or consisting essentially of a compound of the invention that specifically competes for binding to CD38 with CD 31. The invention further relates to the use of a compound disclosed herein that specifically competes for binding to CD38 with CD31 for the preparation or manufacture of a medicament.

The present invention also relates to a method of preventing and/or treating a disease in a subject in need thereof, comprising administering to said subject a compound, composition, pharmaceutical composition or medicament described herein that specifically competes for binding to CD38 with CD 31.

The invention also relates to a compound, composition, pharmaceutical composition or medicament according to the invention which specifically competes for binding to CD38 with CD31 for use as a medicament. The present invention also relates to a compound, composition, pharmaceutical composition or medicament according to the present invention specifically competing for binding to CD38 with CD31 for use in the prevention and/or treatment of a disease in a subject in need thereof.

In one embodiment, the disease is associated with, attributed to, or caused by an elevated level of soluble CD38 in the subject. In particular, increased levels of soluble CD38 can be detected in blood samples (including whole blood, plasma, and serum, preferably plasma samples), cerebrospinal fluid samples, and/or muscle biopsies.

In one embodiment, the disease is selected from the group comprising or consisting of: neurodegenerative diseases; a neuroinflammatory disorder; inflammatory diseases; autoimmune diseases; metabolic diseases; eye diseases; age-related diseases and aging; and cancer and metastasis. In one embodiment, the disease is selected from the group comprising or consisting of: neurodegenerative diseases, neuroinflammatory diseases, inflammatory diseases, autoimmune diseases, and combinations thereof.

The present disclosure also encompasses symptoms of the disease.

Examples of neurodegenerative diseases include, but are not limited to: parkinson's disease and related disorders including Parkinson's disease, Parkinson-dementia (Parkinson-dementia), autosomal recessive PARK2 and PARK 6-related Parkinson's disease, atypical Parkinsonism including progressive supranuclear palsy (progressive subangler palsy), corticobasal degeneration syndrome (corticobasal degeneration syndrome), Lewy body dementia (Lewy bodies dementia), multiple system atrophy (multiple system atrophy), melon prostrate Parkinson's disease (guilloupean disorders) and lykingo-bodig disease; motor neuron diseases, including muscular diseasesAmyotrophic lateral sclerosis, frontotemporal dementia, progressive bulbar paralysis, pseudobulbar paralysis, primary lateral sclerosis, progressive muscular atrophy, spinal muscular atrophy, and post-polio syndrome (post-polio syndrome); a neuroinflammatory disorder; alzheimer's disease and related disorders including early stages of alzheimer's disease, mild stages of alzheimer's disease, moderate stages of alzheimer's disease, mild to moderate stages of alzheimer's disease, late stages of alzheimer's disease, mild cognitive impairment, vascular dementia, mixed dementia, Pick's disease, argyrophilic dementia (argyrophilic brain disease), posterior cortical atrophy (stereosr corticoic dementia), west-Korsakoff Syndrome (Wernicke-Korsakoff Syndrome); prion diseases; lysosomal storage diseases; leukodystrophy (leukodystrophy); huntington's Disease; multiple sclerosis; down syndrome (Down syndrome); spinal and bulbar muscular atrophy; HIV-associated neurocognitive disorders; tourette Syndrome (Tourette Syndrome); autosomal dominant spinocerebellar ataxia; friedriich's Ataxia; dentatorubral pallidoluysian atrophia (Dentatorubral pallidoluysian atrophy); myotonic dystrophy (myotonic dystrophy); schizophrenia; age-related memory disorders; autism and autism spectrum disorders (autism spectrum disorders); attention-deficit hyperactivity disorder (attention-deficiency disorder); chronic pain; dementia caused by alcohol; progressive non-fluent aphasia (progressive non-fluent aphasia); semantic dementia (semantic dementia); spastic paraplegia; fibromyalgia (fibromyalgia); post-Lyme disease (post-Lyme disease); neuropathy; withdrawal symptoms; alpers' disease; brain-eye-facial-skeletal syndrome (cerebro-oculo-fascio-skelol syndrome); wilson's disease; cockayne syndrome (Cockayne syndrome); leigh's disease; neurodegeneration with brain iron accumulation (neurogeneration with brain iron accumulation); ocular clonus myoclonus syndrome (opsoclonus myoclonus syndrome); alpha-methylacyl-CoA racemase deficiency (alpha-methylacyl-CoA racemase deficiency); anderman synthesisSyndrome (Andermann syndrome); arts syndrome (Arts syndrome);syndrome (1)syndrome); mitochondrial membrane protein-associated neurodegeneration; pantothenate kinase-associated neurodegeneration; polycystic lipid membranous osteodysplasia with systemic sclerosis (polycystic lipodystrophy) with sclerosing leukoencephalopathy; riboflavin transporter deficiency neuronal disease (riboflavin transporter deficiency neuronopathy); and ataxia telangiectasia (ataxia telangiectasia).

In one embodiment, the present invention relates to an anti-CD 38 antibody designated "clone B08" or "clone D06" or an antigen-binding fragment thereof as described herein for use in the prevention and/or treatment of a neurodegenerative disease in a subject in need thereof.

The terms "neuroinflammatory disorder" and "inflammatory demyelinating disease" are used interchangeably to refer to a disorder in which one or more regions of the brain or spinal cord are inflamed. However, these terms are not fully equivalent, and those skilled in the art will appreciate that demyelination is not necessarily required for neuroinflammatory diseases, although demyelination typically develops after inflammation.

Examples of neuroinflammatory diseases include, but are not limited to, multiple sclerosis; acute disseminated encephalomyelitis (acute disseminated encephalomyelitis); optic neuritis; transverse myelitis (transverse myelitis); neuromyelitis optica (neuromyelolitis optica) and the like. In some cases, a primary condition with secondary neuroinflammation (e.g., traumatic brain injury with secondary neuroinflammation) may be considered a neuroinflammatory disease as it relates to the subject disclosure.

In one embodiment, the present invention relates to an anti-CD 38 antibody designated "clone B08" or an antigen-binding fragment thereof as described herein for use in the prevention and/or treatment of a neuroinflammatory disease in a subject in need thereof.

Examples of inflammatory diseases include, but are not limited to, arthritis (including osteoarthritis, rheumatoid arthritis, spondyloarthropathies (spondyloarthopathies), and psoriatic arthritis); asthma (including allergic asthma, non-allergic asthma, exercise-induced asthma, drug-induced asthma, occupational asthma, and late-stage asthma); inflammatory bowel diseases (including Crohn's Disease, ulcerative colitis, and colitis); inflammatory dermatoses (including psoriasis, allergic dermatitis, and contact hypersensitivity); multiple sclerosis; osteoporosis; tendonitis; allergic disorders (including rhinitis, conjunctivitis, and urticaria); inflammation in response to damage to the host (including injury or infection); transplant rejection; graft versus host disease; sepsis; and systemic lupus erythematosus.

In one embodiment, the invention relates to an anti-CD 38 antibody, designated "clone B08", "clone C05", "clone D06", or "clone D07", or an antigen-binding fragment thereof, as described herein, for use in the prevention and/or treatment of an inflammatory disease in a subject in need thereof.

Examples of autoimmune diseases include, but are not limited to, alopecia areata (alopecia areata); ankylosing spondylitis; arthritis; antiphospholipid syndrome; autoimmune Addison's disease; autoimmune hemolytic anemia; autoimmune inner ear disease (also known as meniere's disease); autoimmune lymphoproliferative syndrome; autoimmune thrombocytopenic purpura; autoimmune hemolytic anemia; autoimmune hepatitis; bechet's disease; crohn's disease; type 1 diabetes (diabetes mellitis type 1); glomerulonephritis; graves' disease; guillain-barre syndrome (Guillain-barre syndrome); inflammatory bowel disease; lupus nephritis; multiple sclerosis; myasthenia gravis (myasthenia gravis); pemphigus (pemphigus); pernicious anemia (pernicious anemia); polyarteritis nodosa (polyarteritis nodosa); multiple incidenceSarcoidosis (polymyositis); primary biliary cirrhosis; psoriasis; the Reynolds phenomenon (Raynaud's phenomenon); rheumatic fever; rheumatoid arthritis; scleroderma; sicca syndrome (A)syndrome); systemic lupus erythematosus; ulcerative colitis; vitiligo; and Wegener's granulomatosis.

Examples of metabolic diseases include, but are not limited to, diabetes (e.g., type 1 diabetes, type 2 diabetes, gestational diabetes); hyperglycemia; insulin resistance; impaired glucose tolerance (impaired glucose tolerance); hyperinsulinemia (hyperinsulinism); diabetic complications; dyslipidemia (dyslipdemia); hypercholesterolemia (hypercholesterolaemia); hypertriglyceridemia (hypertriglyceridemia); HDL hypocholesterolemia; LDL hypercholesterolemia and/or HLD non-cholesterolemia (HLD non-cholesteleomia); VLDL hyperproteinemia; dyslipoproteinemia; apolipoprotein a-1 hypoproteinemia; metabolic syndrome; syndrome X (syndrome X); obesity; non-alcoholic fatty liver disease (NAFLD); non-alcoholic steatohepatitis; and adrenoleukodystrophy (adrenal leukodystrophy).

As used herein, the term "cancer" is intended to encompass primary and secondary cancers, as well as recurrent, metastatic, and/or multi-drug resistant (multi-drug resistant) cancers.

Examples of cancer include, but are not limited to, breast cancer; prostate cancer; lung cancer; ovarian cancer; colorectal cancer; brain cancer. Other non-limiting examples of cancer include cholangiocarcinoma; bladder cancer; brain cancer, including glioblastoma and medulloblastoma; breast cancer; cervical cancer; choriocarcinoma; colon cancer; endometrial cancer; esophageal cancer; gastric cancer; hematological tumors, including acute lymphocytic and myelogenous leukemia; multiple myeloma; aids-related leukemia and adult T-cell leukemia lymphoma; intraepithelial neoplasms (intraepithelial neoplasms) including Bowen's disease and Paget's disease; liver cancer; lung cancer; lymphomas, including hodgkin's disease and lymphocytic lymphoma; neuroblastoma; oral cancer, including squamous cell carcinoma; ovarian cancer, including those derived from epithelial, stromal, germ, and mesenchymal cells; pancreatic cancer; prostate cancer; rectal cancer; sarcomas, including leiomyosarcoma (leiomyosarcoma), rhabdomyosarcoma (rhabdomyosarcoma), liposarcoma, fibrosarcoma, and osteosarcoma; skin cancers including melanoma, Kaposi's sarcoma, basal cell carcinoma (basocellular cancer), and squamous cell carcinoma; testicular cancer, including reproductive tumors, such as seminoma (seminoma), non-seminoma, teratoma, choriocarcinoma; stromal tumors and germ cell tumors; thyroid cancer, including thyroid adenocarcinoma and medullary carcinoma; and renal cancers, including adenocarcinoma and Wilms' tumor. Other examples of cancers include lymphomas, sarcomas, and carcinomas such as fibrosarcoma, myxosarcomas (myxosarcomas), liposarcoma, chondrosarcoma (chondrosarcoma), osteosarcoma, chordoma (chordoma), angiosarcoma, endotheliosarcoma, lymphangiosarcoma, synovioma, mesothelioma, lymphangioendotheliosarcoma (lymphangioendotheliosarcoma), Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic carcinoma, breast carcinoma, ovarian carcinoma, prostate carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary adenocarcinoma, cystadenocarcinoma (cystadenocarinoma), medullary carcinoma, bronchial carcinoma, renal cell carcinoma, liver carcinoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonic carcinoma, wilms' tumor, cervical carcinoma, testicular tumor, lung carcinoma, non-small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, and cell carcinoma, Astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma; leukemias, e.g., acute lymphocytic leukemia and acute myelogenous leukemia (myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia); chronic leukemia (chronic myelogenous (granulocytic) leukemia and chronic lymphocytic leukemia); and polycythemia vera (polycythemia vera), lymphomas (hodgkins and non-hodgkins), multiple myeloma, Waldenstrom's macrobulinemia, and heavy chain.

Examples of ocular diseases include, but are not limited to, age-related macular degeneration, glaucoma, macular edema, diabetic retinopathy (diabetic retinitis), uveitis, allergic conjunctivitis, scleritis, keratitis, keratoconjunctivitis, vernal keratoconjunctivitis, allergic keratoconjunctivitis, cicatricial conjunctivitis, blepharitis, endophthalmitis, retinitis, retinopathy, choroiditis, sjogren's syndrome, and retinal necrosis.

"aging", also known as "organic aging", is manifested by age-related diseases, the incidence of which increases with age. Death by aging means death by age-related disease. Inhibiting or ameliorating aging may delay one, some or most age-related disorders. Slow aging manifests as a delay in age-related diseases. Slow aging is considered a healthy aging.

Examples of age-related diseases include, but are not limited to, benign tumors; cardiovascular diseases such as stroke, atherosclerosis, and hypertension; hemangioma; osteoporosis; insulin resistance and type II diabetes, including diabetic retinopathy and neuropathy; alzheimer's disease; parkinson's disease; age-related macular degeneration; arthritis; seborrheic keratosis; actinic keratosis; photoaging skin; skin spots (skin spots); skin cancer; systemic lupus erythematosus; psoriasis; smooth muscle cell proliferation and intimal thickening following vascular injury; inflammation; arthritis; side effects of chemotherapy; benign prostatic hyperplasia; and less common diseases, with higher incidence in the elderly than in the young.

In one embodiment, the disease is selected from the group comprising or consisting of: amyotrophic lateral sclerosis; parkinson's disease and related disorders; alzheimer's disease and related disorders; huntington's disease; multiple sclerosis; rheumatoid arthritis; systemic lupus erythematosus; diabetes mellitus; obesity; non-alcoholic steatohepatitis; age-related macular degeneration; and glaucoma. In one embodiment, the disease is selected from the group comprising or consisting of: amyotrophic lateral sclerosis; parkinson's disease and related disorders; alzheimer's disease and related disorders; and multiple sclerosis. In some embodiments, the disease is amyotrophic lateral sclerosis. In some embodiments, the disease is multiple sclerosis. In some embodiments, the disease is parkinson's disease and related disorders. In some embodiments, the disease is alzheimer's disease and related disorders.

In one embodiment, the method of the invention is used for delaying aging in the body.

In one embodiment, the compound, composition, pharmaceutical composition or medicament of the present invention specifically competes for binding to CD38 with CD31 for delaying aging in the body.

The present invention also relates to a method of increasing the level of at least one anti-inflammatory cytokine in a subject in need thereof, in particular in the blood of said subject, comprising administering to said subject a compound, composition, pharmaceutical composition or medicament according to the present invention that specifically competes for binding to CD38 with CD 31.

The present invention also relates to a compound, composition, pharmaceutical composition or medicament according to the present invention specifically competing for the binding of CD38 with CD31 for use in increasing the level of at least one anti-inflammatory cytokine in a subject in need thereof, in particular in the blood of said subject.

Examples of anti-inflammatory cytokines include, but are not limited to, interleukin-10 (IL-10), transforming growth factor beta (TGF-beta), interleukin-1 ra (IL-1ra), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-11 (IL-11), interleukin-13 (IL-13), and interleukin-22 (IL-22).

Methods of measuring blood cytokine levels in a subject are known in the art. Such methods include, for example, detection using ELISA, such as the methods described in the examples section below.

In particular, the present invention relates to a method of increasing IL-10 levels in the blood of a subject in need thereof, especially said subject, comprising administering to said subject a compound, composition, pharmaceutical composition or medicament according to the present invention that specifically competes for binding to CD38 with CD 31.

In particular, the present invention relates to a compound, composition, pharmaceutical composition or medicament according to the present invention specifically competing for the binding of CD38 with CD31 for use in increasing the level of IL-10 in the blood of a subject in need thereof, especially said subject.

Methods of measuring IL-10 levels in a blood sample from a subject are well known in the art and include, for example, using ELISA assays.

In one embodiment, a compound, composition, pharmaceutical composition or medicament described herein that specifically competes for binding to CD38 with CD31 causes an increase in IL-10 levels in the blood of a subject by at least 100%, 200%, 300%, particularly at least 400% and more particularly at least 500% as compared to administration of a negative control molecule.

In one embodiment, the subject is an animal. In one embodiment, the subject is a mammal.

Examples of mammals include, but are not limited to, primates (including humans and non-humans), cows (including cows), horses, pigs, sheep, goats, dogs, and cats.

In a preferred embodiment, the subject is a human.

In one embodiment, the subject is an adult (e.g., a human older than 18 years of age or after acquisition of reproductive capacity). In another embodiment, the subject is a child (e.g., a human being under the age of 18 years or a subject who has not acquired reproductive capacity).

In one embodiment, the subject is male. In one embodiment, the subject is a female.

In one embodiment, the subject is/has been diagnosed with a disease selected from the group comprising or consisting of: neurodegenerative diseases; a neuroinflammatory disorder; inflammatory diseases; autoimmune diseases; metabolic diseases; eye diseases; age-related diseases; and cancer and metastasis.

In one embodiment, the subject is/has been diagnosed with a disease selected from the group comprising or consisting of: amyotrophic lateral sclerosis; parkinson's disease and related disorders; alzheimer's disease and related disorders; huntington's disease; multiple sclerosis; rheumatoid arthritis; systemic lupus erythematosus; diabetes mellitus; obesity; non-alcoholic steatohepatitis; age-related macular degeneration; and glaucoma.

In one embodiment, the subject is at risk of developing a disease selected from the group comprising or consisting of: neurodegenerative diseases; a neuroinflammatory disorder; inflammatory diseases; autoimmune diseases; metabolic diseases; eye diseases; age-related diseases; and cancer and metastasis.

In one embodiment, the subject is at risk of developing a disease selected from the group comprising or consisting of: amyotrophic lateral sclerosis; parkinson's disease and related disorders; alzheimer's disease and related disorders; huntington's disease; multiple sclerosis; rheumatoid arthritis; systemic lupus erythematosus; diabetes mellitus; obesity; non-alcoholic steatohepatitis; age-related macular degeneration; and glaucoma.

In one embodiment, the subject is/has been diagnosed as having an increased level of plasma soluble CD 38.

In one embodiment, the subject is/has been diagnosed as having an increased level of cerebrospinal fluid soluble CD 38.

In one embodiment, the subject is/has been diagnosed with an increase in soluble CD38 levels in a muscle biopsy.

Methods for detecting and measuring the level of soluble CD38 are well known to those skilled in the art. In particular, tools (means) and kits for detecting and measuring the level of soluble CD38 are commercially available.

In one embodiment, a compound, composition, pharmaceutical composition or medicament described herein will be formulated for administration to a subject.

In one embodiment, the compound, composition, pharmaceutical composition or medicament of the present invention will be administered systemically or locally.

In one embodiment, the compound, composition, pharmaceutical composition or medicament described herein will be administered by injection, orally, topically, nasally, buccally, rectally, vaginally, intratracheally, endoscopically, transmucosally or by transdermal administration.

In one embodiment, the compound, composition, pharmaceutical composition or medicament of the invention is to be injected, preferably systemically.

Examples of formulations suitable for injection include, but are not limited to, solutions, e.g., sterile aqueous solutions, gels, dispersions, emulsions, suspensions, solid forms suitable for addition of a liquid prior to use to prepare a solution or suspension, e.g., powders, liposomal forms, and the like.

Examples of systemic injections include, but are not limited to, intravenous (iv), subcutaneous, intramuscular (im), intradermal (id), intraperitoneal (ip), perimedullary injection, and perfusion (perfusion).

In one embodiment, the compound, composition, pharmaceutical composition or medicament of the present invention is sterile when injected. Methods for obtaining sterile compositions include, but are not limited to, GMP synthesis (where GMP stands for "Good manufacturing practice").

Sterile injectable forms of the compositions can be aqueous or oleaginous suspensions. These suspensions may be formulated according to the techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent. Acceptable vehicles and solvents that may be employed include water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono-or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives, are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersing agents, which are commonly used in the formulation of pharmaceutically acceptable dosage forms, including emulsions and suspensions. Other commonly used surfactants, such as Tween, Span and other emulsifiers or bioavailability enhancers, are commonly used to prepare pharmaceutically acceptable solid, liquid or other dosage forms, and may also be used for formulation purposes.

It is to be understood that other suitable routes of administration are also contemplated by the present invention, and the manner of administration will ultimately be determined by the attending physician within the scope of sound medical judgment. In addition to administration by injection (iv, ip, im, etc.), other routes are also available, such as nebulization (Resloud et al 2014.MAbs.6(5): 1347-55; Guilleminault et al 2014.J Control Release.196: 344-54; Resloud et al 2015.Expert Opin Drug Deliv.12(6):1027-39) or subcutaneous administration (Jackisch et al 2014. Gebutshilffe Frauenheilkd.74(4): 343-) 349; Solal-Celigny 2015.Expert Rev Hematol.8(2): 147-53).

In one embodiment, the compound, composition, pharmaceutical composition or medicament of the invention is capable of crossing the Blood Brain Barrier (BBB), preferably when an intravenous or subcutaneous route of administration is used.

Methods of delivering a compound, composition, pharmaceutical composition, or medicament across the blood-brain barrier upon intravenous injection are known in the art, particularly when the compound is an antibody. In fact, several publications indicate that intravenous injection of monoclonal antibodies can effectively cross the Blood Brain Barrier (BBB). Pharmacokinetic modeling of intravenous monoclonal antibody brain concentrations showed that plasma mAb concentrations of about 0.4% were found in the brain (Shah & Bets,2013.MAbs.5(2): 297-305). Brain/plasma ratios of adocamumab (Aducanumab) and ABBV-8E12 were found to reach 1.3% and 0.385, respectively, in humans (Sevigny et al 2016.Nature.537(7618): 50-6; West et al 2017.J Prev Alz Dis.4(4): 236-241). In addition, various strategies have been developed to improve BBB penetration (BBB cross), including the use of bispecific antibodies targeting either the insulin receptor or the transferrin receptor (for review see Neves et al, 2016.Trends Biotech.34(1):36-48) or the use of ultrasound-induced BBB patency (BBB openning), as described in Konofagou et al, 2012.Curr Pharm Biotechnol.13(7): 1332-45.

In one embodiment, a compound, composition, pharmaceutical composition or medicament described herein will be administered to a subject in need thereof in a therapeutically effective amount.

Such therapeutic amounts can be determined by one of skill in the art by routine experimentation, including assessing the effect of the administration of the compound, composition, pharmaceutical composition or medicament on the disease for which prevention and/or treatment is sought by administration of the compound, composition, pharmaceutical composition or medicament described herein.

Such tests can be carried out, for example, by analyzing the quantitative and qualitative effect of the administration of different amounts of the above-mentioned compounds, compositions, pharmaceutical compositions or medicaments according to the invention on a panel of markers (biological and/or clinical) characteristic of the disease, in particular in a biological sample from a subject.

It will be understood, however, that the total daily amount of a compound, composition, pharmaceutical composition or medicament of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disease being treated and the severity of the disease; the activity of the compound, composition, pharmaceutical composition or medicament used; the age, weight, general health, sex, and diet of the subject; the time of administration, route of administration and rate of excretion of the compound, composition, pharmaceutical composition or medicament used; the duration of the treatment; drugs used in combination or concomitantly with the compounds, compositions, pharmaceutical compositions or medicaments used; and other factors well known in the medical arts. For example, it is well known in the art to start doses of the compound at levels below those required to achieve the desired therapeutic effect and to gradually increase the dose until the desired effect is achieved. The total dose required for each treatment may be administered in multiple doses or in a single dose.

In one embodiment, a therapeutically effective amount of a compound, composition, pharmaceutical composition or medicament of the present invention ranges from about 0.1mg/kg to about 50mg/kg, from about 0.5mg/kg to about 45mg/kg, from about 1mg/kg to about 40mg/kg, from about 2.5mg/kg to about 35mg/kg, from about 5mg/kg to about 30mg/kg, from about 10mg/kg to about 20 mg/kg. In one embodiment, a therapeutically effective amount of a compound, composition, pharmaceutical composition or medicament of the present invention is about 15 mg/kg.

In one embodiment, a therapeutically effective amount of a compound, composition, pharmaceutical composition, or medicament described herein ranges from about 10 μ g/kg to about 4mg/kg (human equivalent dose, HED), from about 40 μ g/kg to about 3.6mg/kg (HED), from about 80 μ g/kg to about 3.2mg/kg (HED), from about 200 μ g/kg to about 2.8mg/kg (HED), from about 400 μ g/kg to about 2.4mg/kg (HED), from about 800 μ g/kg to about 2mg/kg (HED). In one embodiment, a therapeutically effective amount of a compound, composition, pharmaceutical composition, or medicament described herein is about 1.2mg/kg (hed).

In one embodiment, a therapeutically effective amount of a compound, composition, pharmaceutical composition, or medicament described herein will be administered once a day, twice a day, three times a day, or more times a day.

In one embodiment, a therapeutically effective amount of a compound, composition, pharmaceutical composition, or medicament described herein will be administered daily, every two days, every three days, every four days, every five days, every six days.

In one embodiment, a therapeutically effective amount of a compound, composition, pharmaceutical composition, or medicament described herein will be administered weekly, biweekly, or every three weeks.

In one embodiment, a therapeutically effective amount of a compound, composition, pharmaceutical composition, or medicament described herein will be administered monthly, bimonthly, trimonthly, quadrate monthly, fifty monthly, or sixty monthly.

In a preferred embodiment, a therapeutically effective amount of a compound, composition, pharmaceutical composition or medicament according to the present invention will be administered every 12 hours, every 24 hours, every 36 hours, every 48 hours, every 60 hours, every 72 hours, every 96 hours.

In one embodiment, the compound, composition, pharmaceutical composition or medicament of the present invention is for acute administration. In one embodiment, the compound, composition, pharmaceutical composition or medicament of the present invention is for long-term administration.

In one embodiment, a therapeutically effective amount of a compound, composition, pharmaceutical composition, or medicament described herein will be administered for about 5 days, 7 days, 10 days, 14 days, 21 days, 28 days, 1 month, 2 months, 3 months, 6 months, 1 year, or longer.

In one embodiment, the compound, composition, pharmaceutical composition or medicament of the present invention is administered before, simultaneously with or after the therapeutic agent.

Some examples of therapeutic agents suitable for co-administration with the compounds, compositions, pharmaceutical compositions, or medicaments described herein include, but are not limited to, immunosuppressive agents, cytotoxins, chemotherapeutic agents, cytokines, immunostimulants, lytic peptides, and radioisotopes.

One skilled in the art will appreciate that the co-administration of a compound, composition, pharmaceutical composition or medicament described herein with a particular therapeutic agent, which may be selected from those described herein without limitation, will depend on the disease or condition to be prevented and/or treated.

Examples of immunosuppressive agents include, but are not limited to, mTOR inhibitors such as sirolimus (sirolimus), everolimus (everolimus), ridaforolimus (ridaforolimus), temsirolimus (temsirolimus), umomolimus (umirolimus), and zotarolimus (zotarolimus); IL-1 receptor antagonists, such as anakinra (anakinra); antimetabolites such as azathioprine (azathioprine), leflunomide (leflunomide), methotrexate (methotrexate), mycophenolic acid, and teriflunomide (teriflunomide); imids such as aplite (apremilast), lenalidomide (lenalidomide), pomalidomide (pomidomide), and thalidomide (thalidomide); and antibodies, such as eculizumab (eculizumab), adalimumab (adalimumab), aflumumab (afleimomab), aphidiimab (afelimomab), certolizumab (certolizumab pegol), golimumab (golimumab), infliximab (infliximab), netilmimab (nerelimomab), meperizumab (mepolizumab), omalizumab (omalizumab), faradalimumab (faramimab), isrimumab (elsimomab), lekulimumab (lebrikizumab), ubulin (usekinumab), securituzumab (secukinuzumab), lomavizumab-CD 3 (muromonoma-CD 3), oitemuzumab (eloxizumab), rituzumab (tepulizumab), vimlizumab (vituzumab), moralizumab (CD 3 (muromonoma-CD 3), oizumab (rituximab), eculizumab (ertuzumab), eculizumab (tiplizumab), edalizumab (ertucizumab), edalizumab (edalizumab), edalizumab (edalizumab), agolizumab (edalizumab), agolizumab), gerbil (edalizumab), galizumab), or (edalizumab), or a, Luxizumab (lumiximab), tenebriximab (teneliximab), toralizumab, aselizumab (aselizumab), galiximab (galiximab), gavimomab (gavilimob), lulizumab (ruplizumab), belimumab (belimumab), Blisimod, ipilimumab (ipilimumab), tiximumab (tremelimumab), tremelimumab (tremelimumab), tielimumab (tremelimumab), netuzumab (natelizumab), tositumumab (tocilizumab), obimumab (bertilimumab), ledellimumab (lerdellimumab), merimab (metulimab), natalizumab (natalizumab), toclizumab (tocilizumab), oxumumab (oduimumab), basiliximab (basiliximab), daclizumab (idalizumab), limolizumab (solizumab), zolizumab (zolizumab), orilizumab (arizumab), orilizumab (rivolizumab), azulizumab (zelizumab), orilizumab (grilizumab), orilizumab (adolizumab), orilizumab (adolizumab), orilizumab (adolizumab), orilizumab (adolizumab), orilizumab (adolizumab), orilizumab (adolizumab), orilizumab (adolizumab), orilizumab (adolizumab), orilizumab (adolizumab), orilizumab (adolizumab), or (ado, Amitocumab (telimomab aritox), valliximab (vapaliximab), vepamumab (vepalimomab), abasic (abatacept), belicept (belatacept), etanercept (etanercept), pernicicept (pegsunecept), aflibercept (aflibercept), alfaxicept (alefacecept) and rilonecept (rilonacept).

Examples of cytotoxins include, but are not limited to, radionuclides (e.g., radionuclides)³⁵S、¹⁴C、³²P、¹²⁵I、¹³¹I、⁹⁰Y、⁸⁹Zr、²⁰¹Tl、¹⁸⁶Re、¹⁸⁸Re、⁵⁷Cu、²¹³Bi and²¹¹at), conjugated radionuclides, and chemotherapeutic agents. Further examples of cytotoxins include, but are not limited to, antimetabolites (e.g., 5-fluorouracil (5-FU), Methotrexate (MTX), fludarabine (fludarabine), and the like), antimicrotubule agents (e.g., vincristine (vincristine), vinblastine (vinblastine), colchicine (colchicine), taxanes (e.g., paclitaxel and docetaxel), and the like), alkylating agents (e.g., cyclophosphamide, melphalan (melphalan), dichloroethylnitrosourea (BCNU), and the like), platinum drugs (e.g., cisplatin (also known as cDDP), carboplatin (carboplatin), oxaliplatin (oxaliplatin), JM-216, CI-973, and the like), anthracyclines (e.g., doxorubicin, daunorubicin, and the like), antibiotics (e.g., mitomycin C), topoisomerase inhibitors (e.g., etoposide), teniposide (JM-973), and other cytotoxic agents (camptothecins), such as ricin (ricin), Diphtheria Toxin (DT), Pseudomonas Exotoxin (PE) A, PE40, abrin (abrin), saporin (saporin), pokeweed viral protein (pokeweed viral protein), ethidium bromide, glucocorticoid (glucoorticoid), anthrax toxin (anthracax toxin), and others.

Examples of chemotherapeutic agents include, but are not limited to, platinum coordination compounds (e.g., cisplatin, carboplatin, or oxaliplatin); taxane compounds (e.g., paclitaxel or docetaxel); topoisomerase I inhibitors (e.g., irinotecan (irinotecan) or topotecan (topotecan)); topoisomerase II inhibitors (e.g., etoposide or teniposide); vinca alkaloids (e.g., vinblastine, vincristine, or vinorelbine); an anti-tumor nucleoside derivative (e.g., 5-fluorouracil, gemcitabine (gemcitabine), or capecitabine); alkylating agents (e.g. nitrogen mustard or nitrosourea, cyclophosphamide, chlorambucil (chlorembucil), carmustine (carmustine) or lomustine (lomustine)), antineoplastic anthracyclines (e.g. daunorubicin, doxorubicin, idarubicin (idarubicin) or mitoxantrone (mitoxantrone)), anti-HER 2 antibodies (e.g. trastuzumab), estrogen receptor antagonists or selective estrogen receptor modulators (e.g. tamoxifen (tamoxifen), toremifene (toremifene), droloxifene (droloxifene), fulvestrant (faslodex) or raloxifene (raloxifene)), aromatase inhibitors (e.g. exemestane (exemestrane), anastrozole (anastrozole), letrozole (letrozole) or letrozole (vorozolone)), retinoids (e.g. retinoids), retinoids (e.g. retinoic acid inhibitors (retinoids), retinoids (e.g. retinol inhibitors (e.g. retinoids), retinoids (e.g. retinol-A, retinoids (retinoids), retinoids (e.g. retinoids), retinol (e.g. retinoids), retinoids (e.g. retinoids), retinoids (e.g. for example, retinoids), retinoids (e.g. for example, retinoids (retinoids), retinoids (e.g. for example, retinoids), retinoids (e.g. for example, retinol (e, retinol (e.g. for example, retinol (e, retinol (e.g. for example, retinol (e, retinol) or (e, retinol) for example, retinol (e, retinol (e.g. for example, retinol (e, retinol (e.g. for example, retinol) for example, retinol (e.g. for example, retinol (, Imatinib mesylate (imatinib mesylate) or gefitinib (gefitinib)); farnesyl transferase inhibitors (farnesyl transferase inhibitors); and HDAC inhibitors.

Examples of cytokines include, but are not limited to, chemokines (e.g., CCL/MCP, CCL/MIP alpha, CCL/MIP beta, CCL/RANTES, CCL, CXCL/IL, CXCL, XCL, and XCL), tumor necrosis factors (e.g., TNFA, Lymphotoxin (Lymphotoxin), TNFSF/CD 40, TNFSF13, and interleukin (e.g., IL-1 beta, TNSF-5, IL-5, and IL-5, CCL, CXCL, CX, IL-7, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36 α, IL-36 β, IL-36 γ, IL-36Ra, IL-37, IL-38, IFN α, IFN β, IFN κ, IFN ω, and GM-CSF).

Examples of immunostimulants include, but are not limited to, filgrastim (filgrastim), pegfilgrastim (pegfilgrastim), legrostastim (lenograstim), morastim (molgrastim), sargrastim (sargramostim), ancestan (ancetim), albicostaton, interferon alpha, peginterferon alpha, interferon beta, peginterferon beta, interferon gamma, aldesleukin (aldesleukin), olprin interleukin (oprevekin), growth hormone, immunocyanine (immunocyanin), pergamase (pegemasin), prolactin (prolactin), tasonamine (tasonermin), histamine dihydrochloride (histamine dihydorzide), poly iclc (iclc), vitamin D, lentinan (lentinan), thymosin (myricetin), mevinosin (myricetin), thymosin (inosine), thymostatin (inosine (1- α), inosine (inosine), and thymostatin (inosine), and cysteine (inosine) peptides (1- α, inosine (inosine, a peptide, a-1-beta-D, a peptide (inosine, a peptide, a-beta, Pidotimod (pidotimod), Bacillus Calmette-guerin vaccine (Bacillus Calmette-guerin vaccine), melanoma vaccine (melanoma vaccine) and sipuleucel-T vaccine.

Examples of lytic peptides include, but are not limited to, toxins (e.g., diphtheria toxin or pseudomonas exotoxin).

Examples of radioisotopes include, but are not limited to, the following radionuclides: technetium (e.g., Tc-99 and Tc-97), potassium (e.g., K-40), rubidium (e.g., Rb-82), iodine (e.g., I-123, I-124, I-125, I-129, I-131), cesium (e.g., Cs-135, Cs-137), cobalt (e.g., Co-60), palladium (e.g., Pd-103, Pd-107), cadmium (e.g., Cd-113), strontium (e.g., Sr-89, Sr-90), europium (e.g., Eu-55), tin (e.g., Sn-121, Sn-126), phosphorus (e.g., P-32, P-33), thallium (e.g., Tl-201), indium (e.g., In-111), gallium (e.g., Ga-67, Ga-68), yttrium (e.g., Y-90), iridium (e.g., Ir-192), bismuth (e.g., Bi-213), Radium (e.g., Ra-223, Ra-225) and ruthenium (e.g., Ru-106).

The present invention also relates to a method of preparing a compound according to the present invention, comprising the step of selecting a compound that specifically competes with CD31, as detailed above.

In one embodiment, where the compound is an antibody, antigen-binding fragment thereof, or antigen-binding antibody mimetic, the method comprises or consists of: selecting K for CD38_DA value of less than or equal to 10^-7M, preferably less than or equal to 10^-8M, more preferably less than or equal to 10^-9M, even more preferably lower than or equal to 1.10^-10The antibody, antigen-binding fragment thereof or antigen-binding antibody mimetic of M can be determined by biosensor analysis, in particular by Biacore analysis.

In one embodiment where the compound is a small organic molecule, the method comprises or consists of: is selected to be less than or equal to 10^-6M, preferably less than or equal to 10^-7M, more preferably less than or equal to 1.10^-8K of M to CD38_DThe value for the replacement of CD31 bound small organic molecules can be determined by biosensor analysis, in particular by Biacore analysis.

In one embodiment wherein the compound is an oligonucleotide, the method comprises or consists of: selecting a K for CD38 of less than or equal to 200nM, preferably less than or equal to 150nM, more preferably less than or equal to 100nM_DThe value for CD 31-bound oligonucleotides can be determined by biosensor analysis, in particular by Biacore analysis.

As described above, the ability of a compound to compete with CD31 can be detected by FACS or Biacore analysis.

In one embodiment where the compound is an antibody, antigen-binding fragment thereof, or antigen-binding antibody mimetic, the method of making a compound of the invention may further comprise the step of selecting an antibody, antigen-binding fragment thereof, or antigen-binding antibody mimetic that specifically binds to at least one epitope within CD38, as defined above.

Preferably, the step of selecting an antibody, antigen-binding fragment thereof or antigen-binding antibody mimetic that specifically binds to at least one epitope within CD38 comprises selecting an antibody, antigen-binding fragment thereof or antigen-binding antibody mimetic that specifically binds to:

-at least one epitope extending at least across amino acid residue 220 to amino acid residue 285 of human CD38 having SEQ ID No. 2; and/or

-cysteine 254 and/or 275 of human CD38 having SEQ ID No. 2; and/or

The 5 th C-terminal disulfide loop comprising cysteine 254 and cysteine 275 of human CD38 having SEQ ID NO 2.

In one embodiment where the compound is an antibody, antigen-binding fragment thereof, or antigen-binding antibody mimetic, the method of making a compound described herein may further comprise the step of selecting an antibody, antigen-binding fragment thereof, or antigen-binding antibody mimetic that does not mediate, trigger, or enhance ADCC, ADCP, and/or CDC.

In one embodiment, the method comprises selecting or consists of a compound having at least one, preferably at least two, more preferably three of the following properties:

it protects dopaminergic neurons from energy deficiency due to inhibition of mitochondrial complex I, in particular mitochondrial neurotoxin MPP⁺；

It uses human Peripheral Blood Mononuclear Cells (PBMCs) to increase the in vitro release of the anti-inflammatory cytokine interleukin-10 (IL-10); and/or

It increases the level of IL-10 in vivo when injected into mice.

In one embodiment, the method comprises or consists of selecting a compound that competes with CD31, preferably with human CD31, and induces tyrosine phosphorylation. In particular, the method comprises selecting for, or consists of, a compound that induces tyrosine phosphorylation of a discrete cytoplasmic matrix that is inhibited in the presence of genistein.

In one embodiment, the method comprises or consists of selecting a compound that competes with CD31, preferably with human CD31, and triggers CD38 internalization.

In one embodiment, the method comprises or consists of selecting a compound that competes with CD31, preferably with human CD31, and induces lysosomal exocytosis. In particular, the method comprises, or consists of, selecting a compound that induces lysosomal exocytosis that is inhibited in the presence of vacuolin-1.

In one aspect, the invention also relates to a method of identifying an individual in need of treatment for a disease selected from the group comprising or consisting of: neurodegenerative diseases, neuroinflammatory diseases, inflammatory diseases, autoimmune diseases, metabolic diseases, ocular diseases, age-related diseases and aging, as well as cancer and metastasis, said subject being susceptible to an effective amount of a compound of the invention, in particular an anti-CD 38 antibody or antigen-binding fragment thereof, said method comprising measuring CD38 levels.

The invention further relates to the use of measuring the level of CD38 as a biomarker in an individual in need of treatment for a disease selected from the group comprising neurodegenerative diseases, neuroinflammatory diseases, inflammatory diseases, autoimmune diseases, metabolic diseases, ocular diseases, age-related diseases and aging, and cancer and metastasis or a combination thereof, in particular to determine whether said individual is susceptible to an effective amount of a compound of the invention, in particular an effective amount of an anti-CD 38 antibody or antigen-binding fragment thereof of the invention.

In one embodiment, the level of CD38 is a plasma CD38 level. In one embodiment, the level of CD38 is a cerebrospinal fluid CD38 level. Methods of measuring CD38 levels are well known in the art and may include ELISA assays using suitable anti-CD 38 antibodies. Suitable antibodies for ELISA can be selected from, for example, R&DAnd (6) purchasing.

In some embodiments, the method further comprises the step of comparing the level of CD38 measured in the individual to a reference value. In one embodiment, the reference value may represent the level of CD38 in a healthy individual or group of healthy individuals. As used herein, "healthy individual" refers to an individual without any of the above-mentioned diseases. In one embodiment, a statistically significant increase in the level of CD38 measured in the individual compared to a reference value can indicate that the individual is susceptible to responding to an anti-CD 38 antibody or antigen-binding fragment thereof of the invention in the treatment of disease.

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