Antagonist anti-CD7 antibodies

Abstract

This invention relates to differentiation cluster 7 (CD7) antagonists, such as antibodies and fragments, as well as methods, uses, and combinations thereof.

Description

Technical Field

[0001] The present invention relates to depletion of cells expressing cluster of differentiation 7 (CD7) or CD7 antagonism, such as antibodies and fragments, as well as methods, uses and combinations. Background Art

[0002] CD7 is a type I transmembrane protein expressed on the surface of T and NK lineage cells. CD7 is highly expressed in T-ALL cells. CD7 has been shown to be an effective biomarker for diagnosing T-ALL. In addition, it is expressed in all CEBPA double-mutated AML cases, including 5-14% of AML patients. It is also expressed in a subset of MDS (myelodysplastic syndrome) primitive cells. Based on these observations, it is expected to target CD7 with ligands such as antibodies to kill tumor cells to treat these malignant diseases.

[0003] T-ALL is a rare and aggressive leukemia that arises from the malignant transformation of T-cell progenitors. Under the current standard of care in multicenter clinical trials, the incidence of relapsed and refractory T-ALL is 20% in children and 40% in adults. Among relapsed patients, only 5% survive more than 5 years. There is clearly a high medical need for those T-ALL patients who are treatment resistant or relapse after initial treatment. Summary of the invention

[0004] In the first configuration , the present invention provides:

[0005] An antibody or fragment comprising a binding site that specifically binds to CD7 (cluster of differentiation 7), wherein the binding site comprises a VH domain encoded by a nucleotide sequence derived from the recombination of a human VH gene segment, a DH gene segment, and a JH gene segment, wherein the VH gene segment is selected from IGHV3-15 and IGHV3-23.

[0006] In the second configuration , the present invention provides:

[0007] An antibody or fragment that specifically binds to CD7 and comprises a VH domain, wherein the VH domain comprises a CDRH3 sequence of an antibody selected from G09, F05, C02 and E04, or the sequence comprising 3, 2 or 1 amino acid substitutions.

[0008] In the third configuration , the present invention provides:

[0009] An antibody or fragment (optionally according to any one of the preceding claims) which specifically binds to CD7 and comprises a VL domain comprising a CDRL3 sequence selected from SEQ ID NO: 13, 16, 33, 36, 53, 56, 73 and 76, or the selected CDRL3 sequence comprising 3, 2 or 1 amino acid substitutions.

[0010] In the fourth configuration , the present invention provides:

[0011] An antibody or fragment comprising a binding site that specifically binds to CD7, wherein the binding site comprises a VL domain, and the VL domain comprises the amino acid sequence of the VL domain of an antibody selected from G09, F05, C02 and E04; or an amino acid that is at least 70% identical thereto.

[0012] In the fifth configuration , the present invention provides:

[0013] An antibody or fragment that specifically binds to CD7 and comprises a heavy chain amino acid sequence of an antibody selected from the group consisting of G09, F05, C02 and E04; or an amino acid that is at least 70% identical thereto.

[0014] In the sixth configuration , the present invention provides:

[0015] An antibody or fragment that specifically binds to CD7 and comprises a light chain amino acid sequence of an antibody selected from the group consisting of G09, F05, C02 and E04; or an amino acid at least 70% identical thereto.

[0016] In the seventh configuration , the present invention provides:

[0017] An antibody or fragment which specifically binds to an epitope of human CD7 which is the same as the epitope bound by the antibody according to any one of the preceding claims.

[0018] In the eighth configuration , the present invention provides:

[0019] An antibody or fragment that competes with the antibody according to any one of the aforementioned configurations for binding to human CD7.

[0020] In the ninth configuration , the present invention provides:

[0021] A combination of an amount of an anti-CD7 antibody or fragment of the invention and an amount of a chemotherapeutic agent.

[0022] In the tenth configuration , the present invention provides:

[0023] A use of the antibody, fragment or combination of the invention in the manufacture of a medicament for administration to a subject to treat or prevent a CD7-mediated disease or condition, optionally cancer.

[0024] In the eleventh configuration , the present invention provides:

[0025] A method of treating or preventing a CD7-mediated disease or condition (optionally cancer) in a subject, the method comprising administering to the subject a therapeutically effective amount of an antibody, fragment or combination of the invention, wherein the CD7-mediated disease or condition is thereby treated or prevented.

[0026] In the twelfth configuration , the present invention provides:

[0027] A pharmaceutical composition comprising the antibody, fragment or combination.

[0028] In the thirteenth configuration , the present invention provides:

[0029] A nucleic acid encoding the VH domain and / or VL domain of the antibody or fragment of the invention.

[0030] In the fourteenth configuration , the present invention provides:

[0031] A nucleic acid encoding: a VH domain comprising the amino acid sequence of the VH domain of an antibody selected from the group consisting of G09, F05, C02 and E04; or an amino acid at least 70% identical thereto.

[0032] In the fifteenth configuration , the present invention provides:

[0033] A nucleic acid encoding: a VL domain comprising the amino acid sequence of the VL domain of an antibody selected from the group consisting of G09, F05, C02 and E04; or an amino acid at least 70% identical thereto.

[0034] In the sixteenth configuration , the present invention provides:

[0035] A nucleic acid comprising

[0036] (a) a nucleotide sequence that is at least 70% identical to the sequence of SEQ ID NO: 10; and / or

[0037] (b) a nucleotide sequence that is at least 70% identical to the sequence of SEQ ID NO:20.

[0038] In the seventeenth configuration , the present invention provides:

[0039] A nucleic acid encoding the heavy chain and / or light chain of an antibody or fragment of the invention.

[0040] In the eighteenth configuration , the present invention provides:

[0041] A nucleic acid encoding a heavy chain comprising an amino acid sequence at least 70% identical to SEQ ID NO:8.

[0042] In the nineteenth configuration , the present invention provides:

[0043] A nucleic acid encoding a light chain comprising an amino acid sequence at least 70% identical to SEQ ID NO:18.

[0044] In the twentieth configuration , the present invention provides:

[0045] A nucleic acid (e.g., in a host cell, e.g., a CHO or HEK293 or Cos cell) comprising

[0046] (a) a nucleotide sequence that is at least 70% identical to a selected heavy chain sequence of an antibody selected from G09, F05, C02 and E04; and / or

[0047] (b) a nucleotide sequence that is at least 70% identical to a selected sequence of an antibody selected from the group consisting of G09, F05, C02, and E04.

[0048] In the twenty-first configuration , the present invention provides:

[0049] A vector comprising the nucleic acid; optionally wherein the vector is a CHO or HEK293 vector.

[0050] In the twenty-second configuration , the present invention provides:

[0051] A host cell comprising the nucleic acid or vector of the present invention.

[0052] In the twenty-third configuration , the present invention provides:

[0053] An antibody, fragment, combination, vector, host cell, use or method as described herein.

[0054] In the twenty-fourth configuration , the present invention provides:

[0055] A method of diagnosing a CD7-mediated disease or condition (optionally cancer) in a subject, the method comprising combining an antibody or fragment of the invention with an isolated cell sample (e.g., a blood or serum sample) and determining that cells comprised by the sample are specifically bound by the antibody or fragment.

[0056] Also provided:

[0057] An in vitro assay for detecting CD7-positive cells in a sample, the assay comprising combining an antibody or fragment of the invention with an isolated cell sample (eg, a blood or serum sample) and determining that cells comprised by the sample are specifically bound by the antibody or fragment. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 : Effect of IgG1 Fc variants on the CDC activity of the anti-CD7 benchmark monoclonal antibody RFT2. CEM cells were plated at 1,750 cells / well in 384-well plates and human complement serum was added at a final concentration of 1 / 16. A titration of each RFT2 IgG1 variant as well as an isotype control was added and incubated at 37°C for 2 hours. The luminescence was read on an Envision plate reader. The percent killing of each antibody was calculated based on the signal from wells without antibody and with / without cells. Error bars represent the standard deviation of 3 replicates for each point. The effective and maximal killing induced by RFT2 E345R was significantly greater than that of the other variants. Data were analyzed using GraphPad Prism v7.02, where the logarithmic antibody concentration (M) was plotted against % killing (mean ± SD), and a 4-parameter logistic curve fit was applied to the data, allowing calculation of the EC 50 Statistical comparisons of the antibodies are shown in Table 6. RFT is discussed in: J Immunol 1989 Dec 1;143(11):3589-97, "Characterization of a human T cell-specific chimeric antibody (CD7) with human constant and mouse variable regions", Heinrich G et al.

[0059] Figure 2 : Flowchart of antibody discovery

[0060] Figure 3 : Secondary screening of antibodies binding to CEM cells and recombinant cynomolgus CD7 CHO cells. Supernatants from hybridoma clones were collected for screening using flow cytometry to detect binding to CD7 expressed in CEM cells or recombinant CHO cells. Binding is represented by geometric mean.

[0061] Figure 4: Characterization of mAbs binding to human and cynomolgus monkey CD7 proteins. Binding was measured by SPR. Single concentration sensorgrams of captured mAbs interacting with human (h) or cynomolgus monkey (c) CD7 proteins as indicated.

[0062] Figure 5 : mAbs were subjected to tertiary screening in CDC assays. Antibodies were run in duplicate for each concentration point and in 3 independent assays. The area under the curve was used to compare antibody potency. 1741E04 E345R and 1741G09 E345R showed the highest CEM cell killing potency, followed by RFT2 E345R, TH69 E345R, 1730C02 E345R, 1896A03E345R, 1734F05 E345R, 1738807 E345R, while isotype controls (IgG1 E345R) and 1734E10 E345R did not show significant efficacy in this assay. Data were analyzed using GraphPad Prism v7.02, where log antibody concentration (M) was plotted against % killing (mean ± SD), and a 4-parameter logistic curve fit was applied to the data, allowing calculation of EC 50 Statistical comparisons of the antibodies are shown in Table 6. TH69 is discussed in: Br J Haematol. 1996 Nov;95(2):327-38, "Therapy with CD7 monoclonal antibody TH-69 is highly effective for xenografted human T-cell ALL", Baum W et al.

[0063] Figure 6 : Tertiary screening of mAbs in ADCP assays. CellTrace TMADCP of monocyte-derived macrophages labeled with violet (CTV) on CFSE-labeled CEM cells pre-conditioned on ice with different concentrations of E345R lead antibody (1730C02, 1734F05, 1741G09) or benchmark anti-CD7 antibody (RFT2) was compared with IgG1 isotype control. CTV-labeled macrophages were co-cultured with CFSE-labeled CEM cells for 2.5 hours. Cells were collected, viability was assessed using a fixable amine-reactive near infrared, and analyzed by flow cytometry. Tumor cell phagocytosis was detected by flow cytometry and expressed as phagocytosis % (phagocytosis % = 100 × (CFSE+ / CTV+%) / (CFSE+%)). The data shown are the mean ± SD of replicate samples from a representative experiment of two independent replicates.

[0064] Figure 7: CDC activity and in vivo pharmacokinetics of 1741G09 E345R and 1741G09 E430G. (A) Two antibodies 1741G09 E345R and 1741G09 E430G and their isotype controls were tested in Figure 6 Both molecules showed similar potent depletion activity (maximum killing and EC 50 ). Error bars represent the standard deviation of 3 replicates for each point. (B) Both antibodies were administered to NODS CIDγ (NSG) mice at a single intravenous dose of 10 mg / kg. Serum samples were collected at eight time points, with three mice taken at each time point and two time points taken for each mouse. The method used to quantify antibody concentration was antigen capture ELISA. Each experimental run consisted of an antibody calibration curve and two sets of QC samples, which were run in duplicate and placed on the front and back of the plate. Antibody concentrations were calculated using SoftMax Pro7, with the standard curve using four-parameter logarithms and 1 / Y weighting. Pharmacokinetic parameters were calculated using the PKSolver Excel plug-in. Based on an unpaired t-test, the P value for the half-life difference was equal to 0.0118. The data shown are triplicate time points (from three different mice). The data are shown as mean concentrations with standard deviations. 1741 G09 430G's t 1 / 2 It is about 7 times longer than 1741G09 345R (20.9 hours vs. 136.7 hours), and the C of G09 430G max Approximately 3 times higher than G09 345R (216811 ng / mL vs. 75001 ng / mL).

[0065] Figure 8: Potent CDC activity of 1741G09 E430G against non-relapsed and relapsed T-ALL cell lines. T-ALL cells including cell lines (Figure 8A), non-relapsed cells derived from patients (Figure 8B), and relapsed cells derived from patients (Figure 8C) were used as target cells and human serum as a complement source. Titration of 1741G09 E430G and isotype controls were added and incubated at 37°C for 2 hours. Add The luminescence was read on an Envision plate reader. The percent killing of each antibody was calculated based on the signal from wells without antibody and with / without cells. Error bars represent the standard deviation of 4 replicates for each point. Data were analyzed using GraphPad Prism v7.02, where log antibody concentration (M) was plotted against % killing (mean ± standard deviation), and a 4-parameter logistic curve fit was applied to the data, allowing calculation of the EC 50 value.

[0066] Fig. 9 : Potent ADCP activity of 1741G09 against relapsed T-ALL cell lines. Monocyte-derived macrophages labeled with violet (CTV) were pre-opsonized with anti-CD7 antibody 1741G09 E430G or appropriate IgG1 isotype control antibody. Phagocytosis of CFSE-labeled CEM or patient T-ALL cells. The 1741G09E430G anti-CD7 antibody induced a concentration-dependent enhancement of CEM and pediatric patient T-ALL (PDTALL-39, -46, -47), and to a lesser extent, adult relapsed T-ALL (PDTALL-Ad2R) phagocytosis. Cell phagocytosis of pediatric patient T-ALL using the 1714G09 E340G antibody was statistically significant compared to isotype control (p=0.015 using paired t-test), and 90-95% of maximum phagocytosis was achieved compared to approximately 70% of maximum phagocytosis of PDTALL-Ad2R cells. Data shown are mean ± SD of samples analyzed in duplicate.

[0067] Fig.10: Cytokine release distribution The ability of 1741G09 E430G antibody to stimulate human PBMCs from five individual donors was assessed, as measured by the release of specific cytokines and chemokines. The corresponding isotype control (IgG1E430G) (A) was used to monitor nonspecific activation of PBMC cultures. After air-drying and fixation on tissue culture plates, super-agonistic anti-CD28 and anti-CD3 (clone OKT3) antibodies were used as positive controls. Human PBMCs were inoculated into the immobilized test reagent in a pre-prepared plate and incubated at 37°C for 48 hours. After incubation, cytokine levels from the culture were measured by Luminex. The induced level of each cytokine was interpolated from the standard curve using a 5-point nonlinear regression analysis. The interpolated data were then normalized to the unstimulated control.

[0068] Fig.11 : PBMC T and NK cell depletion distribution of 1741G09 E430G in CDC assay. Human T (A) or NK (B) cells from two healthy donors were isolated from cryopreserved PBMC using a pan-human T or NK cell isolation kit (Miltenyi Biotech), respectively, and plated at 1,750 cells / well. A titration of 1741G09E430G and a matched isotype control were added and incubated at 4°C for 30 minutes to allow antibody conditioning. Human complement serum was then added at a final concentration of 1 / 16, and the plate was incubated at 37°C for 2 hours. After incubation, Add to all wells, and read the luminescent output on the Envision plate reader. The luminescent signal is related to the number of viable cells per well, and the killing percentage of each antibody concentration is calculated based on the luminescent signal from wells without antibody (0% killing) and without cells (100% killing). Data were analyzed using GraphPad Prism v7.02, where the logarithmic antibody concentration (M) is plotted against the killing percentage (mean ± standard deviation), and a 4-parameter logistic curve fit is applied to the data, allowing calculation of the EC 50 value.

[0069] Fig.12 : Survival of B cells, monocytes, NK cells, and T cells in human whole blood assays. Whole blood from three healthy donors (A, B, C) collected in hirudin tubes was treated with different antibodies in three independent experiments and then incubated at 37°C for 20 hours. Hirudin was used as an anticoagulant to preserve complement activity. After incubation, immunophenotype was analyzed by flow cytometry based on cell surface markers (T: CD45 + CD3 + CD19 - ; B: CD45 + CD3- CD19 + NK: CD45 + CD3 - CD16 + CD56 + ; Monocytes: CD14 + ). Data sets for each donor with mean values ​​(-) are shown. NC': (negative control); IC': isotype control; OFA: Ofatumumab; RTX: Rituximab. Mean values ​​were compared by paired t-test. * refers to p values ​​below 0.05; ** refers to p values ​​below 0.01.

[0070] Fig.13 : Survival rate of CEM cells in blood of healthy donors. Whole blood from three healthy donors (D0457, D0462, and D0463) in hirudin tubes was used CEM cells were spiked with violet (CTV) and treated with 10 μg / mL of antibodies as indicated in three independent experiments. Hirudin was used as an anticoagulant to preserve complement activity. After incubation at 37°C for 20 hours, the immunophenotype was analyzed by flow cytometry based on cell surface markers (T: CD45 + CD3 + CD19 - ; B: CD45 + CD3 - CD19 + ;NK;CD45 + CD3 - CD16 + CD56 + ; Monocytes: CD14 + ). Data sets for each donor with mean values ​​(-) are shown. IC′: isotype control; OFA: ofatumumab. Changes in cell counts compared to isotype control were also determined and plotted in the right graph. Each point represents a single donor, and the mean of 3 donors per condition is represented by a common line. 50% and 90% thresholds are indicated by discontinuous lines. Mean values ​​were compared by paired t-test. * refers to p-values ​​below 0.05; ** refers to p-values ​​below 0.01; *** refers to p-values ​​below 0.001; **** refers to p-values ​​below 0.0001.

[0071] Fig.14: Effect of anti-C5a Ab on 1741G09 E430G-induced cell death (n=1). CTV-labeled CEM cells were spiked with 10 μg / mL of E430G KY1007, E430G IgG1, WT KY1007, or ofatumumab ± 10 μg / mL of anti-C5a Ab in healthy donor blood and incubated at 37°C for 20 hours. A similar method as in the previous section was used in this assay.

[0072] Fig.15 : 1741G09 E430G prolonged the survival rate of xenograft mice. NSG mice were injected with 5×10 6 T-ALL cells, PDTALL46, and then from the 3rd day until the end of the study, 10 mg / Kg was administered three times a week. Two groups (10 mice / group) were treated with 1741G09 E430G and the corresponding isotype control, respectively. (A) Kaplan Meier shows the animals remaining in the study in these two groups. ***: p < 0.0001 Log-Rank (Mantel-Cox) test compared to isotype control. (B) Individual animal flow cytometry data shows the percentage of cells expressing human CD5 on the cell surface present in the blood of the study mice.

[0073] Fig.16 : Complement activity during chemotherapy (see reference 15).

[0074] Fig.17 : CD7 and CRP expression profiles in relapsed T-ALL cell lines, CEMs, and peripheral T and NK cells

[0075] Fig.18 : Complement activation pathway DETAILED DESCRIPTION

[0076] definition

[0077] Unless otherwise defined herein, scientific and technical terms shall have the meanings commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0078] Unless the context clearly indicates otherwise, the singular terms "a", "an", and "the" include plural referents. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. Although methods and materials similar or equivalent to those described herein can be used to practice or test the present disclosure, suitable methods and materials are described below. The abbreviation "eg" is derived from the Latin exempli gratia and is used herein to indicate a non-limiting example. Therefore, the abbreviation "eg" is synonymous with the term "for example".

[0079] In the specification and claims, the term "about" is used to modify values ​​such as the amount of ingredients in the composition, concentration, volume, processing temperature, processing time, yield, flow rate, pressure, etc., and ranges thereof, used in describing the embodiments of the present disclosure. The term "about" refers to the numerical quantity changes that may occur, for example, by typical measurement and processing procedures for preparing compounds, compositions, concentrates, or using formulations; by inadvertent errors in these procedures; by differences in the manufacture, source, or purity of the starting materials or ingredients used to implement the method, and similar approximate considerations. The term "about" also covers amounts that vary due to aging of formulations with specific starting concentrations or mixtures and amounts that vary due to mixing or processing formulations with specific starting concentrations or mixtures. When modified with the term "about", the attached claims include equivalents of these amounts.

[0080] As used herein, "administer" or "administration" refers to the act of injecting or otherwise physically delivering a substance (e.g., an anti-hCD7 antibody provided herein) that is present outside the body into a patient, such as by mucosal, intradermal, intravenous, intramuscular delivery, and / or any other physical delivery method described herein or known in the art. When treating a disease or its symptoms, administration of the substance generally occurs after the onset of the disease or its symptoms. When preventing a disease or its symptoms, administration of the substance generally occurs before the onset of the disease or its symptoms.

[0081] The terms "antibody", "immunoglobulin" or "Ig" may be used interchangeably herein and mean an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or a combination thereof, through at least one antigen recognition site within the variable region of the immunoglobulin molecule. As used herein, the term "antibody" encompasses intact polyclonal antibodies, intact monoclonal antibodies, antibody fragments (such as Fab, Fab', F(ab')2, and Fv fragments), single-chain Fv (scFv) mutants, multispecific antibodies such as bispecific antibodies (including dual binding antibodies), chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising an antigenic determinant portion of an antibody, and any other modified immunoglobulin molecule comprising an antigen recognition site, as long as the antibody exhibits the desired biological activity. The term "antibody" may also refer to a Y-shaped glycoprotein with a molecular weight of approximately 150 kDa made of four polypeptide chains: two light (L) chains and two heavy (H) chains. There are five types of mammalian Ig heavy chain isotypes, represented by the Greek letters alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ). The type of heavy chain defines the class of the antibody, namely IgA, IgD, IgE, IgG, and IgM, respectively. The gamma and alpha classes are further divided into subclasses based on differences in constant domain sequence and function, such as IgGl, hIgG2, mIgG2A, mIgG2B, IgG3, IgG4, IgA1, and IgA2. In mammals, there are two types of immunoglobulin light chains, lambda and kappa. The "variable region" or "variable domain" of an antibody refers to the amino terminal domain of the heavy or light chain of an antibody. The variable domains of the heavy and light chains may be referred to as "V H ” and “V L ". These domains are generally the most variable parts of an antibody (relative to other antibodies of the same class) and contain the antigen binding site. An example of an antibody is a heavy chain only (i.e., H2) antibody, which includes a dimer of heavy chains (5'-VH-(optional hinge)-CH2-CH3-3') and lacks light chains.

[0082] The antibodies described herein can be oligoclonal, polyclonal, monoclonal (including full-length monoclonal antibodies), camelized, chimeric, CDR-grafted, multispecific, bispecific (including bi-binding antibodies), catalytic, chimeric, humanized, fully human, anti-idiotypic, including antibodies that can be labeled in soluble or conjugated form and fragments, variants or derivatives thereof, alone or in combination with other amino acid sequences provided by known techniques. The antibodies can be from any species. The antibodies described herein can be naked or conjugated to other molecules, such as toxins, radioisotopes, etc.

[0083] The terms "antigen binding site", "antigen binding domain", "antigen binding region", "antigen binding fragment" and similar terms refer to the portion of an antibody (e.g., the complementarity determining region (CDR)) that includes the amino acid residues that interact with an antigen and confer specificity and affinity to the binding agent for the antigen. The antigen binding region can be derived from any animal species, such as rodents (e.g., rabbits, rats, or hamsters) and humans. Preferably, the antigen binding region will be of human origin.

[0084] The antigen-binding fragments described herein may include single-chain Fv (scFv), single-chain antibodies, single-domain antibodies, domain antibodies, Fv fragments, Fab fragments, F(ab') fragments, F(ab')2 fragments, antibody fragments exhibiting desired biological activity, disulfide-stabilized variable regions (dsFv), dimeric variable regions (diabodies), anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies directed against antibodies), in vivo antibodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed by antibody fragments and epitope-binding fragments in any of the above. Specifically, the antibodies and antibody fragments described herein may include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules containing antigen-binding sites. Digestion of antibodies with the enzyme papain produces two identical antigen-binding fragments (also referred to as "Fab" fragments) and "Fc" fragments that have no antigen-binding activity but have the ability to crystallize. "Fab" as used herein refers to a fragment of an antibody comprising one constant domain and one variable domain of each of the heavy and light chains. The term "Fc region" herein is used to define the C-terminal region of the immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions. "Fc fragment" refers to the carboxyl terminal portion of the two H chains linked together by disulfide bonds. The effector function of the antibody is determined by the sequence in the Fc region, which is also recognized by Fc receptors (FcR) found on certain types of cells. Digestion of antibodies with the enzyme, pepsin, produces a F(ab')2 fragment in which the two arms of the antibody molecule remain connected and include two antigen binding sites. The F(ab')2 fragment has the ability to cross-link antigens.

[0085] The term "derived from the recombination of..." in relation to gene segments will be apparent to the skilled person, who will understand that B cells reorganize their variable region gene segments to produce the coding sequence of the variable domain. For example, "derived from the recombination of human VH gene segments, DH gene segments and JH gene segments" relates to the recombination of a human VH gene segment together with a DH gene segment and a JH gene segment to form a rearranged VDJ sequence encoding the heavy chain antibody variable domain. Joining and somatic hypermutation may also be features of the process, with the resulting recombinant VDJ sequence comprising one or more nucleotide additions, substitutions or deletions (e.g., p-additions and / or n-additions) not included in the germline V, D and J sequences. For the Vκ and Jκ gene segments of the κ light chain variable domain, and for the Vλ and Jλ of the λ light chain variable domain, it will be said to be equivalent. It is intended that any post-translational modification may be additionally encompassed in the variable domain.

[0086] "Fv" as used herein refers to the smallest fragment of an antibody that retains both the antigen recognition site and the antigen binding site. This region consists of a dimer of a heavy chain variable domain and a light chain variable domain in tight, non-covalent or covalent association. It is in this configuration that the three CDRs of each variable domain interact to define the V H -V L The antigen binding site on the surface of the dimer. The six CDRs together give the antibody antigen binding specificity. However, even a single variable domain (or half of an Fv that includes only three CDRs specific for an antigen) has the ability to recognize and bind to an antigen, although with a lower affinity than the complete binding site.

[0087] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, i.e., the individual antibodies comprising the population are identical, except for possible naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation) that may be present in small amounts. Monoclonal antibodies are highly specific and are directed against a single antigenic determinant or epitope. In contrast, polyclonal antibody preparations typically contain different antibodies directed against different antigenic determinants (or epitopes). As used herein, the term "monoclonal antibody" encompasses both complete monoclonal antibodies and full-length monoclonal antibodies, as well as antibody fragments (e.g., Fab, Fab', F(ab')2, Fv), single-chain (scFv) mutants, including antibody portions, fusion proteins, and any other modified immunoglobulin molecules including antigen recognition sites. In addition, "monoclonal antibody" refers to such antibodies prepared in a variety of ways, including but not limited to hybridomas, phage selection, recombinant expression, and transgenic animals.

[0088] The monoclonal antibodies herein may comprise "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and / or light chain is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies that exhibit the desired biological activity.

[0089] The term "humanized antibody" refers to a subset of chimeric antibodies in which "hypervariable regions" from non-human immunoglobulins (donor antibodies) replace residues from hypervariable regions in human immunoglobulins (recipient antibodies). Typically, a humanized antibody will comprise substantially all of the variable domains in at least one and usually two variable domains, in which all or substantially all of the hypervariable loops correspond to those of non-human immunoglobulin sequences, and all or substantially all of the framework regions are those of human immunoglobulin sequences, although the framework regions may contain one or more substitutions that improve antibody properties such as binding affinity, isomerization, immunogenicity, etc.

[0090] The term "bispecific antibody" is intended to include antibodies that have specificity for two target molecules, including but not limited to the following forms: DVD-Ig (see DiGiammarino et al., "DVD-Ig for Bispecific Targeting TM Design and generation of DVD-Ig molecules TM molecules for dual-specific targeting), "Meth. Mo. Biol.", 2012, 889, 145-156, mAb 2 (See WO2008 / 003103, mAb 2format, the description of which is incorporated herein by reference), FIT-Ig (see WO2015 / 103072, the description of the FIT-Ig scaffold is incorporated herein by reference), mAb-dAb, dock and lock, Fab-arm exchange, SEEDbody, triabody, LUZ-Y, Fcab, κλ body, orthogonal Fab, scDiabody-Fc, diabody Fc, tandem scFv-Fc, Fab-scFv-Fc, Fab-scFv, intrabody, BiTE, diabody, DART, TandAb, scDiabody, scDiabody-CH3, diabody-CH3, tripartite, minibody, minibody, TriBi minibody, scFv-CH3 KIH, scFv-CH-CL-scFv, F(ab′)2-scFv, scFv-KIH, Fab-scFv-Fc, tetravalent HCab, ImmTAC, knobs-in-holes, knobs-in-holes with common light chain, knobs-in-holes with common light chain and charge pairs, charge pairs, charge pairs with common light chain, DT-IgG, DutaMab, IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig and zybody. For a review of bispecific formats, see Spiess, C. et al., Mol. Immunol. (2015). In another embodiment, the bispecific molecule comprises an antibody fused to another non-Ig format, such as a T cell receptor binding domain; an immunoglobulin superfamily domain; an agnathous variable lymphocyte receptor; a fibronectin domain (e.g., Adnectin TM ); antibody constant domain (e.g., CH3 domain, such as Fcab TM CH2 and / or CH3, wherein the constant domain is not a functional CH1 domain; scFv; (scFv)2; sc-bifunctional antibody; scFab; centrosomal protein and derived from CTLA-4 (Evibody TM ) of a scaffold; a lipocalin domain; a protein A, such as a Z domain of protein A (e.g., an Affibody TM or SpA); A domain (e.g. Avimer TM or Maxibody TM); heat shock proteins (e.g., epitope binding domains derived from GroEI and GroES); transferrin domains (e.g., trans-isomers); ankyrin repeat proteins (e.g., DARPin TM ); peptide aptamers; C-type lectin domains (e.g. Tetranectin TM ); human γ-crystallin or human ubiquitin (avidin); a PDZ domain; a scorpion toxin; and a Kunitz-type domain of a human protease inhibitor.

[0091] In one embodiment, the bispecific antibody is a mAb 2 mAb 2 Includes V from intact antibodies fused to modified constant regions H and V L The constant region of the mAb has been engineered to form an antigen binding site and is referred to as an "Fcab". 2 The technology behind the format is described in more detail in WO2008 / 003103 and mAb 2 The description of the form is incorporated herein by reference.

[0092] In another embodiment, the bispecific antibody is a "dual binding antibody". As used herein, the term "dual binding antibody" is a bispecific antibody in which both antigen binding domains are formed by VH / VL pairs and comprises FIT-Ig (see WO2015 / 103072, which is incorporated herein by reference), mAb-dAb, docking and locking, Fab-arm exchange, SEEDbody, trifunctional antibody, LUZ-Y, Fcab, κλ body, orthogonal Fab, scDiabody-Fc, bifunctional antibody Fc, tandem scFv-Fc, Fab-scFv-Fc, Fab-scFv, intrabody, BiTE, bifunctional antibody, DART, TandAb, scDiabody, scDiabody-CH3, bifunctional antibody-CH3, triplet , miniantibodies, miniantibodies, scFv-CH3KIH, scFv-CH-CL-scFv, F(ab′)2-scFv, scFv-KIH, Fab-scFv-Fc, tetravalent HCab, ImmTAC, knobs-in-holes, knobs-in-holes with common light chain, knobs-in-holes with common light chain and charge pairs, charge pairs, charge pairs with common light chain, DT-IgG, DutaMab, IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv and scFv4-Ig.

[0093] The term "hypervariable region", "CDR region" or "CDR" refers to the region of the antibody variable domain that is hypervariable in sequence and / or forms structurally defined loops. In general, the antigen binding site of an antibody contains six hypervariable regions: V H Three of them (CDRH1, CDRH2, CDRH3) and V LThe three (CDRL1, CDRL2, CDRL3) of the heavy and light chains of the antibody confer antigen-binding specificity to the antibody. The CDRs can be defined according to the Kabat system (see Kabat, EA et al., 1991, "Sequences of Proteins of Immunological Interest", 5th edition, NIH publication number 91-3242, U.S. Department of Health and Human Services). Other systems can be used to define CDRs, including the system designed by Chothia et al. (see Chothia, C. and Lesk, AM, 1987, "Canonical structures for the hypervariable regions of immunoglobulins", Journal of Molecular Biology (J. Mol. Biol.), 196, 901-917) and the IMGT system (see Lefranc, MP, 1997, "Unique database numbering system for immunogenetic analysis", Immunol. Today, 18, 50). Antibodies typically contain 3 heavy chain CDRs and 3 light chain CDRs. The term one or more CDRs is used herein to refer to one or several of these regions. Those skilled in the art can easily compare different naming systems and determine whether a particular sequence can be defined as a CDR.

[0094] "Human antibody" is an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human and / or has been prepared using any of the techniques for preparing human antibodies and specifically excludes humanized antibodies including non-human antigen binding residues. The term "specifically binds to" refers to a measurable and reproducible interaction of binding between a target and an antibody such as determining whether a target exists in the presence of a heterogeneous population of molecules comprising biomolecules. For example, an antibody that specifically binds to a target (which may be an epitope) is an antibody that binds to this target with greater affinity, activity, greater ease and / or longer duration than it binds to other targets. In one embodiment, the degree to which an antibody binds to an unrelated target is less than about 10% of the degree to which the antibody binds to the target as measured, for example, by radioimmunoassay (RIA).

[0095] Antibodies or fragments thereof that specifically bind to the human CD7 (hCD7) antigen may cross-react with related antigens. Preferably, antibodies or fragments thereof that specifically bind to the hCD7 antigen do not cross-react with other antigens (but may optionally cross-react with CD7 of a different species, such as rhesus monkeys or mice). For example, the antibody or fragment thereof may be cross-reacted by immunoassay, BIAcore TM Or other techniques known to those skilled in the art to identify antibodies or fragments thereof that specifically bind to the hCD7 antigen. As determined using experimental techniques such as radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA), when the affinity of the antibody or its fragment binding to the hCD7 antigen is higher than the affinity of binding to any cross-reactive antigen, the antibody or its fragment specifically binds to the CD7 antigen. Typically, the specific or selective reaction will be at least twice the background signal or noise, and more typically more than 10 times the background (such as more than 15 times, more than 20 times, more than 50 times or more than 100 times). For discussion on antibody specificity, see, for example, Paul, ed., 1989, Fundamental Immunology, Second Edition, Raven Press, New York, pp. 332-336.

[0096] The term "aliphatic amino acid" means that the amino acid R group is non-polar and hydrophobic. The hydrophobicity increases with the number of C atoms in the hydrocarbon chain. Glycine, alanine, valine, leucine and isoleucine are aliphatic amino acids.

[0097] The term "aromatic amino acid" means that the amino acid R group contains an aromatic ring system. Phenylalanine, tyrosine and tryptophan are aromatic amino acids.

[0098] The term "hydroxyl-containing amino acid" means that the amino acid R group contains a hydroxyl group and is hydrophilic. Serine, cysteine, threonine and methionine are hydroxyl-containing amino acids.

[0099] The term "basic amino acid" means that the amino acid R group is nitrogen-containing and is basic at neutral pH. Histidine, lysine, and arginine are basic amino acids.

[0100] The term "cyclic amino acid" means that the amino acid R group has an aliphatic ring structure. Proline is the only cyclic aliphatic amino acid.

[0101] The term "acidic amino acid" means that the amino acid R group is polar and negatively charged at physiological pH. Aspartic acid and glutamic acid are acidic amino acids.

[0102] The term "amide amino acid" means that the amino acid R group contains an amide group. Asparagine and glutamine are amide amino acids.

[0103] As used herein, "authorization number" or "marketing authorization number" refers to the number issued by the regulatory agency when the agency determines that a specific medical product and / or composition can be marketed and / or offered for sale in the area under the jurisdiction of the agency. As used herein, "regulatory agency" refers to one of the agencies responsible for evaluating the safety and efficacy of, for example, medical products and / or compositions and controlling the sales / marketing of such products and / or compositions in a given area. The Food and Drug Administration (FDA) of the U.S. and the European Medicines Agency (EPA) in Europe are just two examples of such regulatory agencies. Other non-limiting examples may include SDA, MPA, MHPRA, IMA, ANMAT, Hong Kong Department of Health Drug Office, CDSCO, New Zealand Medicines Administration (Medsafe) and KFDA.

[0104] As used herein, "buffer" refers to a chemical agent that is able to absorb a certain amount of acid or base without undergoing a drastic change in pH.

[0105] As used herein, the term "carrier" refers to a diluent, adjuvant (e.g., Freund's adjuvant (complete and incomplete)), excipient or vehicle administered with a therapeutic agent. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, plant or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. When the pharmaceutical composition is administered intravenously, water is a preferred carrier. Saline solutions and aqueous solutions of dextrose and glycerol can also be used as liquid carriers, particularly for injectable solutions.

[0106] The term "chemotherapeutic agent" or "chemotherapy" refers to a therapeutic agent whose primary purpose is to destroy cancer cells, usually by interfering with the growth or reproduction ability of tumor cells. There are many different types of chemotherapeutic agents, of which more than 50 approved chemotherapy drugs are available. Chemotherapeutic drugs can be classified based on how they work. Alkylating drugs kill cancer cells by directly attacking the genetic material of genes, DNA. Cyclophosphamide is an alkylating drug. Antimetabolites interfere with the production of DNA and prevent cell growth and reproduction. An example of an antimetabolite is 5-fluorouracil (5-FU). Antitumor antibiotics are made from natural substances such as fungi in soil. Antitumor antibiotics interfere with important cell functions, including the production of DNA and cell proteins. Doxorubicin and bleomycin belong to this group of chemotherapy drugs. Plant alkaloids prevent cells from dividing normally. Vinblastine and vincristine are plant alkaloids obtained from the periwinkle plant. Steroid hormones slow the growth of some cancers that rely on hormones. For example, tamoxifen is used to treat breast cancer that relies on estrogen for growth. DNA damage response (DDR) inhibitors, such as PARP inhibitors, block the DNA repair mechanism after single-strand or double-strand breaks.

[0107] Examples of chemotherapeutic agents include doxorubicin, doxorubicin, 5-fluorouracil, cytarabine (Ara-C), cyclophosphamide, Thiotepa, Taxotere (docetaxel), busulfan, cytotoxins, paclitaxel, methotrexate, cisplatin, melphalan, vinblastine, bleomycin, etoposide, ifosfamide, mitomycin C, mitoxantrone, vincristine, vinorelbine, carboplatin, teniposide, daunomycin, carminomycin, aminopterin, actinomycin, mitomycin, esperamicin (see U.S. Pat. No. 4,675,187), melphalan, and other related nitrogen mustards. Suitable toxins and chemotherapeutic agents are described in Remington's Pharmaceutical Sciences, 19th edition (Mack Publishing Co. 1995), and Goodman and Gilman's The Pharmacological Basis of Therapeutics, 7th edition (MacMillan Publishing Co. 1985). Another example of a chemotherapeutic agent is an antibody-conjugated toxin, including but not limited to pyrrolobenzodiazepines, maytansinoids, calicheamicin, and the like. Other suitable toxins and / or chemotherapeutic agents are known to those skilled in the art.

[0108] As used herein, the term "composition" is intended to encompass a product comprising the specified ingredients (eg, antibodies of the invention), optionally in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients, optionally in the specified amounts.

[0109] As used herein, the terms "comprising" or "comprises" are used when referring to antibodies, fragments, uses, compositions, methods, and their respective components that are essential to the methods or compositions, but remain open to the inclusion of unspecified elements, whether essential or not.

[0110] The term "consisting of" refers to the antibodies, fragments, uses, compositions, methods and corresponding components thereof as described herein, excluding any elements not recited in the description of the embodiments.

[0111] As used herein, the term "consisting essentially of" refers to those elements required for a given embodiment. The term permits the presence of elements that do not materially affect the basic and novel or functional characteristics of the embodiment.

[0112] In the context of polypeptides, as used herein, the term "derivative" encompasses polypeptides that include the amino acid sequence of a hCD7 polypeptide, a fragment of a hCD7 polypeptide, or an antibody or fragment that specifically binds to a hCD7 polypeptide that has been altered by the introduction of amino acid residue substitutions, deletions, or additions. As used herein, the term "derivative" also encompasses hCD7 polypeptides, fragments of hCD7 polypeptides, or antibodies that specifically bind to hCD7 polypeptides that have been chemically modified, such as by covalent attachment of any type of molecule to the polypeptide. For example, but not limited to, hCD7 polypeptides, fragments of hCD7 polypeptides, or hCD7 antibodies may be chemically modified, such as by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, attachment to cellular ligands or other proteins, and the like. Derivatives are modified in a manner that differs from the naturally occurring or starting peptide or polypeptide, whether in the type or position of the attached molecule. Derivatives further encompass the absence of one or more chemical groups that are naturally present on the peptide or polypeptide. Derivatives of hCD7 polypeptides, fragments of hCD7 polypeptides or hCD7 antibodies can be chemically modified by chemical modification using techniques known to those skilled in the art, including but not limited to specific chemical cleavage, acetylation, formulation, metabolic synthesis of tunicamycin, etc. Further, derivatives of hCD7 polypeptides, fragments of hCD7 polypeptides or hCD7 antibodies may contain one or more non-classical amino acids. The polypeptide derivatives have similar or identical functions to the hCD7 polypeptides, fragments of hCD7 polypeptides or hCD7 antibodies described herein.

[0113] As used herein, the term "effector function" (or "effector enabling") refers to one or more of antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC)-mediated responses, Fc-mediated phagocytosis, antibody-dependent cellular phagocytosis (ADCP) or antibody-dependent phagocytosis and antibody recycling through the FcRn receptor.

[0114] "Effective amount" refers to an effective amount measured in dosages and lasting for the required period of time to achieve a desired effect (including treatment or prevention results). "Therapeutically effective amount" refers to the minimum concentration required to achieve a measurable improvement or prevention of a particular condition. The therapeutically effective amount herein can vary according to factors such as the patient's disease state, age, sex, and weight, as well as the ability of the antibody to elicit a desired response in an individual. The therapeutically effective amount is also the amount in which the therapeutic beneficial effects exceed the toxic or deleterious effects of the antibody. "Preventionally effective amount" refers to an effective amount measured in dosages and lasting for the required period of time to achieve a desired prevention result. In some embodiments, the effective amount of an antibody of the present invention is from about 0.1 mg / kg (mg antibody per kg subject body weight) to about 100 mg / kg. In certain embodiments, the effective amount of the antibody provided herein is about 0.1 mg / kg, about 0.5 mg / kg, about 1 mg / kg, 3 mg / kg, 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, about 50 mg / kg, about 60 mg / kg, about 70 mg / kg, about 80 mg / kg, about 90 mg / kg or about 100 mg / kg (or ranges therein). In some embodiments, "effective amount" as used herein also refers to the amount of an antibody of the invention that achieves a specified result (e.g., inhibiting the hCD7 biological activity of a cell).

[0115] As used herein, the term "epitope" refers to a local area on the surface of an antigen (such as an hCD7 polypeptide or hCD7 polypeptide fragment) that is capable of binding to one or more antigen binding regions of an antibody and has antigenic or immunogenic activity that is capable of eliciting an immune response in an animal, preferably a mammal, and most preferably a human. An epitope with immunogenic activity is a portion of a polypeptide that elicits an antibody response in an animal. An epitope with antigenic activity is a portion of a polypeptide to which an antibody specifically binds, as determined by any method known in the art, such as by an immunoassay described herein. An antigenic epitope is not necessarily immunogenic. An epitope is typically composed of chemically active surface groups of molecules such as amino acids or sugar side chains and has specific three-dimensional structural characteristics and specific charge characteristics. The region of a polypeptide that contributes to an epitope may be continuous amino acids of a polypeptide, or the epitope may come from two or more non-continuous regions of a polypeptide together. An epitope may or may not be a three-dimensional surface feature of an antigen. In certain embodiments, an hCD7 epitope is a three-dimensional surface feature of an hCD7 polypeptide (e.g., in the form of a trimer of an hCD7 polypeptide). In other embodiments, the hCD7 epitope is a linear feature of the hCD7 polypeptide (e.g., in a trimer or monomeric form of the hCD7 polypeptide). The antibodies provided herein can specifically bind to an epitope in a monomeric (denatured) form of hCD7, an epitope in a trimer (native) form of hCD7, or both a monomeric (denatured) form and a trimer (native) form of hCD7. In specific embodiments, the antibodies provided herein specifically bind to an epitope in a trimer form of hCD7 but do not specifically bind to a monomeric form of hCD7.

[0116] As used herein, the term "excipient" refers to an inert substance commonly used as a diluent, vehicle, preservative, binder or stabilizer for a drug, and includes, but is not limited to, proteins (e.g., serum albumin, etc.), amino acids (e.g., aspartic acid, glutamic acid, lysine, arginine, glycine, histidine, etc.), fatty acids and phospholipids (e.g., alkyl sulfonates, octanoates, etc.), surfactants (e.g., SDS, polysorbates, nonionic surfactants, etc.), sugars (e.g., sucrose, maltose, trehalose, etc.), and polyols (e.g., mannitol, sorbitol, etc.). See also Remington's Pharmaceutical Sciences (1990) Mark Publishing Company, Easton, Pennsylvania, which is hereby incorporated by reference in its entirety.

[0117] In the context of a peptide or polypeptide, the term "fragment" as used herein refers to a peptide or polypeptide that includes less than the full-length amino acid sequence. Such fragments may be produced, for example, by truncation of the amino terminus, truncation of the carboxyl terminus, and / or internal deletions of one or more residues of the amino acid sequence. For example, a fragment may result from alternative RNA splicing or from in vivo protease activity. In certain embodiments, the CD7 fragment comprises a polypeptide comprising an amino acid sequence of at least 5 consecutive amino acid residues, at least 10 consecutive amino acid residues, at least 15 consecutive amino acid residues, at least 20 consecutive amino acid residues, at least 25 consecutive amino acid residues, at least 40 consecutive amino acid residues, at least 50 consecutive amino acid residues, at least 60 consecutive amino acid residues, at least 70 consecutive amino acid residues, at least 80 consecutive amino acid residues, at least 90 consecutive amino acid residues, at least 100 consecutive amino acid residues, at least 125 consecutive amino acid residues, at least 150 consecutive amino acid residues, at least 175 consecutive amino acid residues, at least 200 consecutive amino acid residues, or at least 250 consecutive amino acid residues of the amino acid sequence of the hCD7 polypeptide or the antibody that specifically binds to the hCD7 antigen. In specific embodiments, the fragment of the hCD7 polypeptide or the antibody that specifically binds to the hCD7 antigen retains at least 1, at least 2, or at least 3 functions of the polypeptide or antibody.

[0118] The term "free" may refer to a polypeptide, such as CD7 or fragments and variants thereof, in combination with a buffer, wherein the polypeptide is not associated with a cell surface or cell membrane. Thus, the term "free" may refer to a polypeptide capable of surface expression (i.e., comprising one or more transmembrane domains or membrane association domains) but not expressed on the surface of a cell or bound to a protein expressed on the surface of a cell in its current state. A free polypeptide may also refer to a free recombinant or natural or unbound polypeptide. In the context of phage display, free antigens may be selected in solution (referred to herein as "soluble selection") or adsorbed to a surface, such as adsorbed to the surface of a 96-well plate (referred to herein as "bio-panning selection").

[0119] As used herein, the term "fusion protein" refers to a polypeptide comprising the amino acid sequence of an antibody and the amino acid sequence of a heterologous polypeptide or protein (i.e., a polypeptide or protein that is not normally a part of an antibody (e.g., a non-anti-CD7 antigen antibody). When used in connection with CD7 or anti-CD7 antibodies, the term "fusion" refers to the connection of a peptide or polypeptide or a fragment, variant and / or derivative thereof to a heterologous peptide or polypeptide. Preferably, the fusion protein retains the biological activity of the CD7 or anti-CD7 antibody. In certain embodiments, the fusion protein comprises a CD7 antibody VH domain, a VL domain, a VH CDR (one, two or three VH CDRs) and / or a VL CDR (one, two or three VL CDRs), wherein the fusion protein specifically binds to a CD7 epitope.

[0120] When used with respect to antibodies, the term "heavy chain" refers to five different types of amino acid sequences of antibodies based on heavy chain constant domains, referred to as α (α), δ (6), ε (ε), γ (γ) and μ (μ). These different types of heavy chains are well known, and produce five antibody classes IgA, IgD, IgE, IgG and IgM, respectively, comprising four subclasses of IgG, i.e. IgG1, IgG2, IgG3 and IgG4. Preferably, the heavy chain is a human heavy chain. In the human population, there are multiple heavy chain constant region alleles of each immunoglobulin or immunoglobulin subclass. The nucleotide and amino acid sequences of these allelic variants can be obtained in publicly available databases such as IMGT, ENSEMBL, Swiss-Prot and Uniprot. Allelic variants can also be identified in various genome sequencing projects. In one embodiment, the antibodies and antibody fragments disclosed herein include heavy chains encoded by IgG1 constant region alleles, including but not limited to human IGHG1*01, IGHG1*02, IGHG1*03, IGHG1*04, and IGHG1*05. In one embodiment, the antibodies and antibody fragments disclosed herein include proteins encoded by IgG2 constant region alleles, including but not limited to human IGHG2*01, IGHG2*02, IGHG2*03, IGHG2*04, IGHG2*05, and IGHG2*06. In one embodiment, the antibodies or antibody fragments disclosed herein include proteins encoded by IgG3 constant region alleles, which include but are not limited to human IGHG3*01, IGHG3*02, IGHG3*03, IGHG3*04, IGHG3*05, IGHG3*06, IGHG3*07, IGHG3*08, IGHG3*09, IGHG3*10, IGHG3*11, IGHG3*12, IGHG3*13, IGHG3*14, IGHG3*15, IGHG3*16, IGHG3*17, IGHG3*18 and IGHG3*19. In one embodiment, the antibodies or antibody fragments disclosed herein include proteins encoded by IgG4 constant region alleles, including but not limited to human IGHG4*01 (see, e.g., the sequence listing herein), IGHG4*02 (see, e.g., the sequence listing herein), IGHG4*03 (see, e.g., the sequence listing herein), and IGHG4*04 (see, e.g., the sequence listing herein). In another example, the heavy chain is an ineffective IgG isotype, such as an ineffective IgG4. In certain embodiments, the antibodies of the present invention include a human γ 4 constant region. In another embodiment, the heavy chain constant region does not bind to the Fc-γ receptor, and, for example, includes a Leu235Glu mutation. In another embodiment, the heavy chain constant region includes a Ser228Pro mutation to increase stability.In another embodiment, the heavy chain constant region is IgG4-PE (see, e.g., the sequence listing herein). In another embodiment, the antibodies and antibody fragments disclosed herein include a heavy chain constant region encoded by a murine IgG1 constant region allele, including but not limited to mouse IGHG1*01 or IGHG1*02. In one embodiment, the antibodies and antibody fragments disclosed herein include a heavy chain constant region encoded by a murine IgG2 constant region allele, including but not limited to mouse IGHG2A*01, IGHG2A*02, IGHG2B*01, IGHG2B*02, IGHG2C*01, IGHG2C*02, or IGHG2C*03. In one embodiment, the antibodies or antibody fragments disclosed herein include a protein encoded by a murine IgG3 constant region allele, including but not limited to mouse IGHG3*01.

[0121] As used herein, the term "host" refers to an animal, preferably a mammal, most preferably a human.

[0122] As used herein, the term "host cell" refers to a particular subject cell transfected with a nucleic acid molecule and the progeny or potential progeny of such a cell. The progeny of such a cell may differ from the parent cell transfected with the nucleic acid molecule due to mutations or environmental influences that may occur in subsequent generations or integration of the nucleic acid molecule into the host cell genome.

[0123] In the context of administering other therapies, the term "combination" refers to the use of more than one therapy. The use of the term "combination" does not limit the order in which the subject with the disease is administered therapy. The subject with a disease mediated by CD7 may be administered the first therapy before (e.g., 1 minute, 45 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks or 12 weeks), simultaneously or thereafter (e.g., 1 minute, 45 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks or 12 weeks). Any additional therapy may be administered in any order with other additional therapies. In certain embodiments, the antibodies of the invention can be administered in combination with one or more therapies (e.g., a therapy that is not an antibody of the invention and is currently administered to prevent, treat, manage and / or ameliorate a CD7-mediated disease). Non-limiting examples of therapies that can be administered in combination with the antibodies of the invention include analgesics, anesthetics, antibiotics, or immunomodulators, or any other agent listed in the U.S. Pharmacopeia and / or Physician's Desk Reference.

[0124] As used herein, an "injection device" refers to a device designed to perform an injection, which includes the step of temporarily fluidly coupling the injection device to a person's tissue, typically subcutaneous tissue. The injection further includes administering a quantity of liquid medication into the tissue and detaching or removing the injection device from the tissue. In some embodiments, the injection device may be an intravenous device or IV device, which is a type of injection device used when the target tissue is blood within the circulatory system, such as blood in a vein. A common but non-limiting example of an injection device is a needle and a syringe.

[0125] As used herein, "instructions" refers to written, printed or graphic material displayed on the immediate container of an article, such as written material displayed on a vial containing a pharmaceutically active agent, or details regarding the composition and use of a product of interest contained in a kit containing a composition of interest. The instructions set forth the intended method of treatment to be administered or performed.

[0126] An "isolated" or "purified" antibody or protein is an antibody or protein that has been identified, separated and / or recovered from a component of its production environment (e.g., natural or recombinant). For example, an antibody or protein is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the antibody is derived, or substantially free of chemical precursors or other chemicals when chemically synthesized. The term "substantially free of cellular material" includes preparations of antibodies in which the antibodies are separated from cellular components of the cells from which the antibodies are isolated or recombinantly produced. Thus, antibodies that are substantially free of cellular material include antibody preparations having less than about 30%, 20%, 10% or 5% (by dry weight) of heterologous proteins (also referred to herein as "contaminating proteins"). When the antibody is recombinantly produced, the antibody is preferably also substantially free of culture medium, i.e., culture medium accounts for less than about 20%, 10% or 5% of the volume of the protein preparation. When the antibody is produced by chemical synthesis, the antibody is preferably substantially free of chemical precursors or other chemicals, i.e., the antibody is separated from chemical precursors or other chemicals involved in the synthesis of the protein. Thus, such antibody preparations have less than about 30%, 20%, 10%, 5% (by dry weight) of chemical precursors or compounds other than the antibody of interest. In preferred embodiments, the antibodies of the invention are isolated or purified.

[0127] The term "Kabat numbering" and similar terms are recognized in the art and refer to a system for numbering amino acid residues that are more variable (i.e., hypervariable) than other amino acid residues in the heavy chain variable region of an antibody or antigen-binding portion thereof (Kabat et al. (1971) Ann. NY Acad. Sci., 190: 382-391 and / or Kabat et al. (1991) Protein Sequences of Immunological Significance, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). For the heavy chain variable region, the hypervariable region is generally within the range of amino acid positions 31 to 35 of CDR1, amino acid positions 50 to 65 of CDR2, and amino acid positions 95 to 102 of CDR3.

[0128] As used herein, "label" or "labeled" refers to the addition of a detectable moiety to a polypeptide, such as a radioactive label, a fluorescent label, an enzymatic label, a chemiluminescent label, or a biotinyl or gold. A radioisotope or radionuclide may comprise 3 H. 14 C. 15 N. 35 S. 90 Y. 99 Tc, 115 In, 125 I. 131 I, fluorescent labels can include rhodamine, lanthanide phosphorus or FITC, and enzymatic labels can include horseradish peroxidase, β-galactosidase, alkaline galactosidase. Additional labels include, by way of illustration and not limitation: enzymes such as glucose-6-phosphate dehydrogenase ("G6PDH"), α-D-galactosidase, glucose oxidase, glucoamylase, carbonic anhydrase, acetylcholinesterase, lysozyme, malate dehydrogenase, and peroxidase; dyes (e.g., cyanine dyes, such as Cy5 TM 、Cy5.5 TM or Cy7 TM); Additional fluorescent labels or fluorescent agents include, for example, fluorescein and its derivatives, fluorescent dyes, GFP (GFP for "green fluorescent protein"), other fluorescent proteins (e.g., mCherry, mTomato), dansyl, umbelliferone, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde, and fluorescamine; fluorophores, such as lanthanide cryptates and chelates, for example, europium, etc. (PerkinElmer Elmer) and Cisbio assays); chemiluminescent labels or chemiluminescent agents, such as isoluminol, luminol and dioxetane; photosensitizers; coenzymes; enzyme substrates; particles, such as latex or carbon particles; metal sols; microcrystals; liposomes; cells that can be further labeled with dyes, catalysts or other detectable groups, etc.; molecules, such as biotin, digoxigenin or 5-bromodeoxyuridine; toxin moieties, for example, selected from Pseudomonas exotoxin (PE or a cytotoxic fragment or mutant thereof), diphtheria toxin or a cytotoxic fragment or mutant thereof, botulinum toxin A, B, C, D, E or F, ricin or a cytotoxic fragment thereof, such as ricin A, abrin or a cytotoxic fragment thereof, saporin or a cytotoxic fragment thereof, pokeweed antiviral toxin or a cytotoxic fragment thereof, and bryophyllin 1 or a cytotoxic fragment thereof.

[0129] When used with respect to antibodies, the term "light chain" refers to immunoglobulin light chains, which exist in mammals in two types, λ (λ) and kappa (κ). Preferably, the light chain is a human light chain. Preferably, the light chain constant region is a human constant region. In the human population, there are multiple light chain constant region alleles. The nucleotide and amino acid sequences of these allele variants can be obtained in publicly available databases such as IMGT, ENSEMBL, Swiss-Prot and Uniprot. In one embodiment, antibodies or antibody fragments disclosed herein include proteins encoded by human kappa constant region alleles, and the constant region alleles include but are not limited to IGKC*01 (see, for example, the sequence table herein), IGKC*02 (see, for example, the sequence table herein), IGKC*03 (see, for example, the sequence table herein), IGKC*04 (see, for example, the sequence table herein) and IGKC*05 (see, for example, the sequence table herein). In one embodiment, the antibodies or antibody fragments disclosed herein include proteins encoded by human lambda constant region alleles, including but not limited to IGLC1*01 (see, e.g., the sequence listing herein), IGLC1*02 (see, e.g., the sequence listing herein), IGLC2*01 (see, e.g., the sequence listing herein), IGLC2*02 (see, e.g., the sequence listing herein), IGLC2*03 (see, e.g., the sequence listing herein), IGLC3*01 (see, e.g., the sequence listing herein), IGLC3*02 (see, e.g., the sequence listing herein), IGLC3*03 (see, e.g., the sequence listing herein), IGLC3*04 (see, e.g., the sequence listing herein), IGLC6*01 (see, e.g., the sequence listing herein), IGLC7*01 (see, e.g., the sequence listing herein), IGLC7*02 (see, e.g., the sequence listing herein), IGLC7*03 (see, e.g., the sequence listing herein). In another embodiment, the antibodies and antibody fragments disclosed herein include a light chain constant region encoded by a mouse kappa constant region allele, including but not limited to IGKC*01, IGKC*03, or IGKC*03. In another embodiment, the antibodies and antibody fragments disclosed herein include a light chain constant region encoded by a mouse lambda constant region allele, including but not limited to IGLC1*01, IGLC2*01, or IGLC3*01.

[0130] "Percent (%) amino acid sequence identity" and "homology" with respect to peptide, polypeptide or antibody sequences are defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in a particular peptide or polypeptide sequence, after aligning the sequences and introducing gaps (if necessary) to achieve the maximum percent sequence identity and not considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be achieved in a variety of ways that are within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEG ALIGN. TM (DNASTAR) software. In one embodiment, the % homology is about 70%. In one embodiment, the % homology is about 75%. In one embodiment, the % homology is about 80%. In one embodiment, the % homology is about 85%. In one embodiment, the % homology is about 90%. In one embodiment, the % homology is about 92%. In one embodiment, the % homology is about 95%. In one embodiment, the % homology is about 97%. In one embodiment, the % homology is about 98%. In one embodiment, the % homology is about 99%. In one embodiment, the % homology is 100%.

[0131] The terms "naturally occurring" or "native" when used in connection with biological materials such as nucleic acid molecules, polypeptides, host cells, etc., refer to those materials found in nature and have not been manipulated by humans.

[0132] As used herein, "packaging" refers to how the components are organized and / or confined into units suitable for distribution and / or use. The packaging may include, for example, boxes, bags, syringes, ampoules, vials, tubes, clamshell packaging, barriers and / or containers to maintain sterility, labeling, etc.

[0133] As used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the US Pharmacopeia, European Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

[0134] As used herein, the terms "polynucleotide," "nucleotide," nucleic acid "nucleic acid molecule," and other similar terms are used interchangeably and include DNA, RNA, mRNA, and the like.

[0135] As used herein, the terms "prevent," "preventing," and "prevention" refer to the complete or partial inhibition of the development, recurrence, onset, or spread of an hCD7-mediated disease and / or symptoms associated therewith, resulting from the administration of a therapy or combination of therapies provided herein (e.g., a combination of prophylactic or therapeutic agents, such as an antibody of the invention).

[0136] The term "soluble" refers to a polypeptide lacking one or more transmembrane or cytoplasmic domains found in a native or membrane-associated form, such as CD7 and variants or fragments thereof. In one embodiment, a "soluble" form of CD7 lacks a transmembrane domain and a cytoplasmic domain.

[0137] The term "subject" or "patient" refers to any animal, including but not limited to mammals. As used herein, the term "mammal" refers to any vertebrate that nurses its young and gives birth to live young (eumammalia or placental mammals) or lays eggs (metatheria or non-placental mammals). Examples of mammalian species include, but are not limited to, humans and other primates, including non-human primates, such as chimpanzees and other apes and monkeys; livestock, such as cattle, sheep, pigs, goats, and horses; domestic mammals, such as dogs and cats; laboratory animals, including rodents, such as mice, rats (including cotton rats), and guinea pigs; birds, including poultry, wild birds, and game birds, such as chickens, turkeys and other gallinaceous birds, ducks, geese, etc.

[0138] As used herein, "substantially all" means at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or about 100%.

[0139] As used herein, the term "therapeutic agent" refers to any agent that can be used to treat, manage, or improve a CD7-mediated disease and / or symptoms associated therewith. In certain embodiments, the term "therapeutic agent" refers to an antibody of the present invention. In certain other embodiments, the term "therapeutic agent" refers to an agent other than an antibody of the present invention. Preferably, the therapeutic agent is an agent that is known to be useful or has been used or is currently used to treat, manage, or improve a CD7-mediated disease or one or more symptoms associated therewith. In a specific embodiment, the therapeutic agent is a fully human anti-CD7 antibody, such as a fully human anti-CD7 monoclonal antibody.

[0140] As used herein, the term "therapy" refers to any regimen, method and / or agent that can be used to prevent, manage, treat and / or improve CD7-mediated diseases (e.g., cancer). In certain embodiments, the terms "therapies" and "therapy" refer to biological therapies, supportive therapies and / or other therapies that can be used to prevent, manage, treat and / or improve CD7-mediated diseases known to those skilled in the art, such as medical personnel.

[0141] The terms "treat," "treatment," and "treating" refer to a reduction or improvement in the progression, severity, and / or duration of an hCD7-mediated disease (e.g., cancer) resulting from the administration of one or more therapies (including but not limited to the administration of one or more prophylactic or therapeutic agents, such as antibodies of the invention).

[0142] The term "variable region" or "variable domain" refers to a part of a light chain and a heavy chain, usually about 120 to 130 amino acids at the amino terminal in the heavy chain, and about 100 to 110 amino acids in the light chain, which differ greatly in sequence between antibodies and are used for the binding and specificity of each specific antibody to its specific antigen. The variability of the sequence is concentrated in those regions referred to as complementary determining regions (CDRs), while the regions with higher conservatism in the variable domains are referred to as framework regions (FRs). The CDRs of CD7 and heavy chains are primarily responsible for the interaction of antibodies with antigens. The numbering of amino acid positions used herein is according to the EU index, as in Kabat et al. (1991) "Protein Sequences with Immunological Significance" (U.S. Department of Health and Human Services, Washington, D.C.) 5th edition ("Kabat et al."). In a preferred embodiment, the variable region is a human variable region.

[0143] Definitions of common terms in cell biology and molecular biology can be found in the following references: The Merck Manual of Diagnosis and Therapy, 19th Edition, published by Merck Research Laboratories, 2006 (ISBN 0-911910-19-0); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); Benjamin Lewin, Genes X, published by Jones & Bartlett Publishing, 2009 (ISBN-10: 0763766321); Kendrew et al. (eds.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference. Reference), VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8, and Current Protocols in Protein Sciences 2009, Wiley Intersciences, Coligan et al., eds.

[0144] Unless otherwise indicated, the present invention was performed using standard procedures as described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual (4th Edition), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA (2012); Davis et al., Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (1995); or Methods in Enzymology: Guide to Molecular Cloning Techniques, Vol. 152, Editors: S.L. Berger and A.R. Kimmel, Academic Press Inc., San Diego, USA (1987); A Guide to Contemporary Protein Science (CPPS) (Editors: John E. Coligan et al., John Wiley and Sons Publishing Company, Inc., San Diego, USA (1987); Sons, Inc.), Current Protocols in Cell Biology (CPCB) (Editors: Juan S. Bonifacino et al., John Wiley & Sons), and Culture of Animal Cells: A Manual of Basic Technique, Author: R. Ian Freshney, Publisher: Wiley-Liss; 5th Edition (2005), Animal Cell Culture Methods (Methods in Cell Biology, Vol. 57, Editors: Jennie P. Mather and David Barnes, Academic Press, 1st Edition, 1998), all of which are incorporated herein by reference in their entirety.

[0145] Additional terms are defined herein within the description of various aspects of the invention.

[0146] Target principle

[0147] CD7 is a 40 kDa transmembrane glycoprotein of the Ig superfamily (1). During early T cell ontogeny, it is expressed in peripheral blood T cells, NK cells, thymocytes, and bone marrow CD34+ CD38 is expressed on the surface of T cells (2). In addition to late memory T cells and effector CD8 + CD7 is expressed on most T cells outside of T cells. CD7 expression has been reported during early T cell development. However, CD7 is not expressed in hematopoietic lineage stem cells (HSCs) (2, 3), suggesting that HSCs are not affected by anti-CD7 antibodies. Although most peripheral T cells are normally CD7 positive, a small percentage of circulating CD4 + Memory cells (CD4 + CD45RA - CD45R0 + ) lacks CD7(4).

[0148] The natural ligand of CD7 has not been identified. CD7 has been shown to act as a co-stimulatory molecule, and anti-CD7 monoclonal antibodies (mAbs) are reported to be mitogenic, increase calcium flux and increase IL-2 production (5). CD7 binds to phosphatidylinositol 3-kinase (PI 3-kinase) and associates with type II PI 4-kinase via a cytoplasmic tyrosine-based YEDM motif (6). However, the exact signal transduction mechanism remains unknown. Although some laboratories have reported relatively minor immune system defects, such as reduced antigen-specific T cell triggering, defective antigen-specific cytotoxic T cell generation, and protection from lipopolysaccharide-induced shock syndrome, no major phenotypic abnormalities have been identified in CD7-deficient mice (7, 8).

[0149] CD7 can be rapidly internalized following antibody binding, as demonstrated on a human T-ALL cell line, CEM cells, where more than 50% of cell surface CD7 was internalized within 30 minutes following antibody attachment (9). In clinical studies with the mouse-human chimeric antibody RFT2, the half-life of the antibody in humans was reported to be approximately 12 hours (10). Following internalization, the intracellular pathways of internalized CD7 are not well described, as it can be recycled to the cell membrane or sent directly to the lysosome for degradation. This rapid internalization may affect the pharmacokinetic and pharmacodynamic profiles of monoclonal antibodies in treated patients.

[0150] CD7 in T-ALL and AML

[0151] CD7 is highly expressed on malignant immature T cells and is usually absent on malignant mature T cells, such as CD4 + Sezary leukemia and HTLV-1 +Adult T-cell leukemia cells (11). In leukemia cells from diagnostic bone marrow samples obtained from patients with T-ALL, the median percentage of CD7 expression was >99% (12). High CD7 expression levels were also observed in samples collected from patients with relapsed T-ALL. CD7 expression levels in leukemia cells at diagnosis or relapse consistently exceeded levels measured in residual normal T cells in the same samples, and standard of care (SoC) chemotherapy did not affect CD7 expression in leukemia cells. In bone marrow samples containing minimal residual disease (MRD) collected during chemotherapy, >99% of the residual leukemia cells were CD7 + Because CD7 expression levels remain high during therapy (12), CD7 is an excellent flow cytometric biomarker for the diagnosis of T-ALL (Table 5).

[0152] In addition to T-ALL, CD7 is expressed on leukemic cells in 15% of acute myeloid leukemia (AML) cases (13). CD7 expression in this subset of AML cases is associated with loss of the wild-type CCAAT / enhancer binding protein alpha (CEBPA) gene due to mutation or silencing of epigenetic mechanisms (14). Such mutational or epigenetic screening in AML has potential clinical importance, allowing for the stratification of subsets of AML for CD7-targeted therapies.

[0153] In conclusion, cell depletion strategies using anti-CD7 monoclonal antibodies hold promise for addressing adult T-ALL and CD7 + AML pathology and other CD7 + Unmet medical needs in cancer.

[0154] Normal T cell development is a tightly regulated process in which T progenitor cells migrate from the bone marrow to the thymus and differentiate into mature and functional T cells. During this differentiation process, dysregulation of oncogenes and tumor suppressor genes can drive immature thymocytes into uncontrolled clonal expansion and lead to T-ALL (22). T-ALL accounts for approximately 20% of all ALL cases and is more common in adults than in children, but the incidence decreases with age. Patients typically present with high white blood cell counts and may also present with organomegaly, especially mediastinal enlargement. Despite improved understanding of T-ALL disease biology, the standard of care (SoC) has remained largely unchanged over the past decade and is still comprised of intensive multi-drug chemotherapy that may be followed by autologous hematopoietic stem cell transplantation. In the case of this SoC, children and adults treated have relapse rates of 20% and 40%, respectively (23, 24). Long-term survival in relapsed and refractory settings is very low, with less than 5% of patients subsequently achieving a second response. Many patients with refractory or relapsed B-cell lymphoblastic leukemia have achieved complete remission and prolonged survival after receiving new-generation targeted therapies (25). However, there has been little new drug development activity for the treatment of T-ALL, likely due to its relatively small patient population size.

[0155] T-ALL malignancies represent a group of hematologic cancers with high rates of relapse and mortality in patients for which effective targeted therapies do not exist. There remains a large unmet medical need to improve clinical outcomes for patients with relapsed and refractory T-ALL.

[0156] Thus, in one aspect, the present invention can be used to treat T-ALL, such as refractory T-ALL.

[0157] Thus, the present invention provides various anti-CD7 antibodies and fragments (such as Fab or scFv fragments), uses and methods. Examples are listed in the following numbered clauses.

[0158] 1. An antibody or fragment comprising a binding site that specifically binds to CD7 (cluster of differentiation 7), wherein the binding site comprises a VH domain encoded by a nucleotide sequence derived from the recombination of a human VH gene segment, a DH gene segment, and a JH gene segment, wherein the VH gene segment is selected from IGHV3-15 and IGHV3-23.

[0159] In an example, the VH gene segment is IGHV3-15*01 or IGHV3-23*04. For example, the DH gene segment and the JH gene segment are human gene segments.

[0160] In an example, as further described below, in KD, K off and / or K onIn an example, specific binding is performed at a KD of 1 pM to 5 nM.

[0161] The skilled artisan is familiar with databases and other sources of antibody gene segments for humans and other species. For example, the IMGT database ( www.IMGT.org ) is a suitable source, such as the version of September 1, 2018.

[0162] With reference to the Examples, antibodies based on IGHV3-15 or IGHV3-23 are shown. Surprisingly, these human VH gene segments produce anti-CD7 antibodies with desirable anti-CD7 properties, such as those described in the Examples.

[0163] 2. The antibody or fragment according to clause 1, wherein the DH gene segment is a human gene segment selected from IGHD3-9, IGHD3-10 and IGH6-19.

[0164] In an example, the DH gene segment is a human gene segment selected from IGHD3-9*01, IGHD3-10*01 and IGH6-19*01.

[0165] 3. An antibody or fragment according to clause 1 or 2, wherein the JH gene segment is a human gene segment selected from IGHJ6, IGHJ4 and IGHJ5.

[0166] In an example, the JH gene segment is a human gene segment selected from IGHJ6*02, IGHJ4*02 and IGHJ5*02.

[0167] 4. The antibody or fragment according to any of the preceding clauses, wherein the binding site comprises a CDRH3 sequence selected from SEQ ID NO: 3, 6, 23, 26, 43, 46, 63 and 66.

[0168] 5. An antibody or fragment according to any of the preceding clauses, wherein the binding site comprises (i) a VH structure of SEQ ID NO: 7 paired with a VL domain comprising SEQ ID NO: 17; (ii) a VH structure comprising SEQ ID NO: 27 paired with a VL domain comprising SEQ ID NO: 37; (iii) a VH structure comprising SEQ ID NO: 47 paired with a VL domain comprising SEQ ID NO: 57; or (iv) a VH domain comprising SEQ ID NO: 67 paired with a VL domain comprising SEQ ID NO: 77.

[0169] 6. The antibody or fragment according to any of the preceding clauses, wherein the binding site comprises a VH domain comprising SEQ ID NO: 7.

[0170] 7. The antibody or fragment according to any of the preceding clauses, wherein the binding site comprises a VH structure of SEQ ID NO: 7 paired with a VL domain comprising SEQ ID NO: 17.

[0171] 8. An antibody or fragment which specifically binds to CD7 and comprises the CDRH3 sequence of the anti-CD7 antibody according to any one of the preceding clauses, or the CDRH3 sequence comprising 3, 2 or 1 amino acid substitutions.

[0172] 9. An antibody or fragment (optionally according to any of the preceding clauses) which specifically binds to CD7 and comprises a VH domain comprising the CDRH3 sequence of an antibody selected from G09, F05, C02 and E04, or the sequence comprising 3, 2 or 1 amino acid substitutions.

[0173] 10. An antibody or fragment according to clause 9, wherein the VH domain comprises (i) a CDRH3 sequence of an antibody selected from G09, F05, C02 and E04; or said CDRH3 sequence comprising 3, 2 or 1 amino acid substitutions; and (ii) a CDRH1 sequence of said selected antibody; or said CDRH1 sequence comprising 3, 2 or 1 amino acid substitutions.

[0174] 11. An antibody or fragment according to clause 9 or 10, wherein the VH domain comprises (iii) a CDRH3 sequence of an antibody selected from G09, F05, C02 and E04; or said CDRH3 sequence comprising 3, 2 or 1 amino acid substitutions; and (iv) a CDRH2 sequence of said selected antibody; or said CDRH2 sequence comprising 3, 2 or 1 amino acid substitutions.

[0175] 12. An antibody or fragment (optionally according to any of the preceding clauses) comprising a binding site that specifically binds to CD7, wherein the binding site comprises a VH domain comprising the amino acid sequence of the VH domain of an antibody selected from G09, F05, C02 and E04; or an amino acid that is at least 70% identical thereto.

[0176] For example, the identity is at least 85%. For example, the identity is at least 90%. For example, the identity is at least 95%.

[0177] 13. The antibody or fragment according to clause 9, 10, 11 or 12, wherein the selected antibody is G09.

[0178] 14. An antibody or fragment according to any of the preceding clauses, comprising a first copy and a second copy of the VH domain.

[0179] In an example, the antibody or fragment comprises a binding site comprising a VH domain of the invention paired with a VL domain of the invention, wherein the binding site is capable of specifically binding to CD7 (e.g., mature CD7, such as human and / or cynomolgus CD7). For example, the antibody or fragment comprises two of such binding sites.

[0180] 15 An antibody or fragment (optionally according to any of the preceding clauses), which comprises a binding site that specifically binds to CD7, wherein the binding site comprises a VL domain encoded by a nucleotide sequence derived from the recombination of a human VL gene segment and a JL gene segment, wherein the VL gene segment is selected from IGKV1D-39, IGKV1-39, IGKV3-11, IGKV1-16 and IGKV1-5.

[0181] In an example, the VL gene segment is selected from IGKV1D-39*01, IGKV1-39*01, IGKV3-11*01, IGKV1-16*02 and IGKV1-5*03; or is selected from IGKV1D-39*01, IGKV3-11*01, IGKV1-16*02 and IGKV1-5*03.

[0182] 16. The antibody or fragment according to clause 15, wherein VL is Vκ and the JL gene segment is a human gene segment selected from IGKJ1 and IGKJ4.

[0183] In an example, VI is Vκ and the JL gene segment is a human gene segment selected from IGKJ1*01 and IGKJ4*01.

[0184] 17. An antibody or fragment which specifically binds to CD7 and comprises the CDRL3 sequence of an anti-CD7 antibody according to any one of the preceding clauses, or the CDRL3 sequence comprising 3, 2 or 1 amino acid substitutions.

[0185] 18 An antibody or fragment (optionally according to any of the preceding clauses) which specifically binds to CD7 and comprises a VL domain comprising a CDRL3 sequence selected from SEQ ID NO: 13, 16, 33, 36, 53, 56, 73 and 76, or the selected CDRL3 sequence comprising 3, 2 or 1 amino acid substitutions.

[0186] 19. An antibody or fragment (optionally according to any of the preceding clauses) that specifically binds to cluster of differentiation 7 (CD7) and comprises a VL domain comprising the CDRL3 (and optionally CDRH3) sequence of an antibody selected from G09, F05, C02 and E04; or said sequences each comprising 3, 2 or 1 amino acid substitutions.

[0187] 20. An antibody or fragment according to clause 19, wherein the VL domain comprises (i) a CDRL3 sequence (and optionally CDRH3) of an antibody selected from G09, F05, C02 and E04; or said CDR3 sequence each comprising 3, 2 or 1 amino acid substitutions; and (ii) a CDRL1 (and optionally CDRH1) sequence of said selected antibody; or said CDR1 sequence each comprising 3, 2 or 1 amino acid substitutions.

[0188] Preferably, the antibody of choice herein is G09 or comprises the variable domain of G09.

[0189] 21. An antibody or fragment according to clause 19 or 20, wherein the VL domain comprises (iii) a CDRL3 (and optionally CDRH3) sequence of an antibody selected from G09, F05, C02 and E04; or said CDR3 sequence each comprising 3, 2 or 1 amino acid substitutions; and (iv) a CDRL2 (and optionally CDRH2) sequence of said selected antibody; or said CDR2 sequence each comprising 3, 2 or 1 amino acid substitutions.

[0190] 22. An antibody or fragment (optionally according to any of the preceding clauses) comprising a binding site that specifically binds to CD7, wherein the binding site comprises a VL domain, the VL domain comprising the amino acid sequence of the VL domain of an antibody selected from G09, F05, C02 and E04; or an amino acid that is at least 70% identical thereto.

[0191] Optionally, an antibody or fragment (optionally according to any of the preceding clauses) is provided, which comprises a binding site that specifically binds to cluster of differentiation 7 (CD7), wherein the binding site comprises a VL domain, the VL domain comprising the amino acid sequence of the VL domain of an antibody selected from G09, F05, C02 and E04; or an amino acid that is at least 70, 80, 85, 90, 95, 96, 97, 98 or 99% identical thereto.

[0192] For example, the identity is at least 85%. For example, the identity is at least 90%. For example, the identity is at least 95%.

[0193] Optionally, the antibody or fragment comprises a first copy and a second copy of the VL domain.

[0194] In an example, the antibody or fragment comprises a binding site comprising a VL domain of the invention paired with a VH domain, wherein the binding site is capable of specifically binding to CD7 (e.g., mature CD7, such as human and / or cynomolgus CD7). For example, the antibody or fragment comprises two of such binding sites.

[0195] 23. An antibody or fragment (optionally according to any of the preceding clauses) that specifically binds to cluster of differentiation 7 (CD7) and comprises the heavy chain amino acid sequence of an antibody selected from G09, F05, C02 and E04; or amino acids that are at least 70, 80, 85, 90, 95, 96, 97, 98 or 99% identical thereto.

[0196] For example, the identity is at least 85%. For example, the identity is at least 90%. For example, the identity is at least 95%.

[0197] 24. An antibody or fragment (optionally according to any of the preceding clauses) that specifically binds to cluster of differentiation 7 (CD7) and comprises the light chain amino acid sequence of an antibody selected from G09, F05, C02 and E04; or amino acids that are at least 70, 80, 85, 90, 95, 96, 97, 98 or 99% identical thereto.

[0198] For example, the identity is at least 85%. For example, the identity is at least 90%. For example, the identity is at least 95%.

[0199] 25. The antibody or fragment according to clause 23, comprising the light chain amino acid sequence of the selected antibody; or amino acids at least 70, 80, 85, 90, 95, 96, 97, 98 or 99% identical thereto.

[0200] For example, the identity is at least 85%. For example, the identity is at least 90%. For example, the identity is at least 95%.

[0201] 26. An antibody or fragment (optionally according to any of the preceding clauses) which specifically binds to an epitope of human CD7 that is the same as the epitope bound by the antibody according to any of the preceding clauses.

[0202] 27. The antibody or fragment according to clause 26, wherein the epitope is identified by unrelated amino acid scanning or by X-ray crystallography.

[0203] The contact amino acid residues involved in the interaction between antibody and antigen can be determined by various methods known to those skilled in the art.

[0204] In one embodiment, the amino acids of the antigen sequence are sequentially substituted (using standard molecular biology techniques to mutate the DNA encoding the antigen sequence), in which case CD7 is substituted with alanine (also known as alanine scanning) or another unrelated amino acid, which may provide residues whose mutations reduce or eliminate the ability of the antibody to recognize the relevant antigen. Binding can be assessed using standard techniques, such as, but not limited to, SPR, HTRF, ELISA (which are described elsewhere herein). Other substitutions can be made to enhance the destruction of binding, such as changing the charge on the side chains of the amino acids of the antigen sequence (e.g., lysine to glutamic acid), converting polar and non-polar residues (e.g., serine to leucine). Alanine scanning or other amino substitution methods can be performed with recombinant soluble antigens, or in the case where the target is a cell membrane target, using transient or stable expression of a mutant version directly on the cell.

[0205] In one embodiment, protein crystallography can be used to determine the contact residues between antibody and antigen (i.e., determine the epitope to which antibody binds), and crystallography allows direct visualization of contact residues involved in antibody-antigen interactions. In addition to standard X-ray crystallography, cryo-electron microscopy has been used to determine the contact residues between antibody and HIV capsid protein (see Lee, Jeong Hyun et al. "Antibodies to a conformational epitope on gp41 neutralize HIV-1 by destabilizing the Env spike.", Nature Communications, 6, (2015)).

[0206] In one embodiment, if the antibody recognizes a linear epitope, short peptides based on the antigen sequence can be generated and standard techniques such as, but not limited to, SPR, HTRF, ELISA (described elsewhere herein) can be used to assess the binding of the antibody to these peptides. By performing alanine scanning on any peptide that shows binding, further studies of the epitope can be provided. As an alternative to linear peptides, conformational scanning can be performed using peptide scanning (Pepscan) technology (http: / / www.pepscan.com / ) using chemical bonds on the peptide to the scaffold, which has been used to determine discontinuous epitopes on CD20 targeting antibodies (Niederfellner, Gerhard et al. "Epitope characterization and crystal structure of GA101 provide insights into the molecular basis for type I / II distinction of CD20 antibodies.", Blood, 118.2, (2011), 358-367).

[0207] In one embodiment, limited proteolytic digestion and mass spectrometry can be used to identify binding epitopes. Antibody-antigen complexes are digested by proteases (such as but not limited to trypsin). The digested composite peptides are compared with independent antibodies and independent antigen digestion mass spectra to determine whether a specific epitope is protected by complex. Further work involving amino acid substitutions and competitive binding can then be adopted to reduce the independent amino acid residues involved in interaction (see, for example, Suckau, Detlev et al. "Molecular epitope identification by limited proteolysis of an immobilized antigen-antibody complex and mass spectrometric peptide mapping by limited proteolysis of immobilized antigen-antibody complex and mass spectrometric peptide mapping. ", Proceedings of the National Academy of Sciences, 87.24, (1990), 9848-9852).

[0208] Thus, in one embodiment, the contact residues of the epitope are identified using an unrelated amino acid scan (e.g., an alanine scan). In another embodiment, an unrelated amino acid scan (e.g., an alanine scan) is performed using a technique selected from a combination of SPR, HTRF, ELISA, X-ray crystallography, cryo-electron microscopy, and limited proteolytic digestion and mass spectrometry. In one embodiment, an unrelated amino acid scan (e.g., an alanine scan) is performed using HTRF. In one embodiment, an unrelated amino acid scan (e.g., an alanine scan) is performed using ELISA.

[0209] When alanine scanning is performed by ELISA or HTRF, if the signal is reduced by at least 25%, the amino acid residue is identified as contributing to the epitope. In one embodiment, the signal is reduced by at least 30%. In one embodiment, the signal is reduced by at least 35%. In one embodiment, the signal is reduced by at least 40%. In one embodiment, the signal is reduced by at least 45%. In one embodiment, the signal is reduced by at least 50%. In one embodiment, the signal is reduced by at least 55%. In one embodiment, the signal is reduced by at least 60%. In one embodiment, the signal is reduced by at least 70%. In one embodiment, the signal is reduced by at least 75%. In one embodiment, the signal is reduced by at least 80%. In one embodiment, the signal is reduced by at least 85%. In one embodiment, the signal is reduced by at least 90%.

[0210] When alanine scanning is performed using SPR, an amino acid residue is identified as contributing to the epitope if the affinity is reduced by at least 10 times. In one embodiment, the affinity is reduced by at least 15 times. In one embodiment, the affinity is reduced by at least 20 times. In one embodiment, the affinity is reduced by at least 30 times. In one embodiment, the affinity is reduced by at least 40 times. In one embodiment, the affinity is reduced by at least 50 times. In one embodiment, the affinity is reduced by at least 100 times.

[0211] In one embodiment, the contact residues of the epitope are identified by X-ray crystallography. In one embodiment, the contact residues of the epitope are identified by cryo-electron microscopy. In one embodiment, the contact residues of the epitope are identified by a combination of limited proteolytic digestion and mass spectrometry.

[0212] 28. An antibody or fragment according to clause 27, wherein the contact residues of the epitope are defined by at least a 10-fold decrease in affinity in an irrelevant amino acid scan, such as an alanine scan determined by SPR.

[0213] In one embodiment, the affinity is reduced by at least 15 times. In one embodiment, the affinity is reduced by at least 20 times. In one embodiment, the affinity is reduced by at least 30 times. In one embodiment, the affinity is reduced by at least 40 times. In one embodiment, the affinity is reduced by at least 50 times. In one embodiment, the affinity is reduced by at least 100 times. SPR can be performed as described herein.

[0214] 29. An antibody or fragment (optionally according to any of the preceding clauses) that competes with the antibody according to any of the preceding clauses for binding to human CD7.

[0215] Optionally, competition is determined by surface plasmon resonance (SPR) or ELISA. For example, the skilled person will be familiar with these techniques and standard conditions.

[0216] In one embodiment, the antibody or fragment competes with hCD7 (or its fusion protein) (e.g., in a dose-dependent manner) for binding to hCD7 expressed on the cell surface. In one embodiment, the antibody or fragment competes with hCD7 (or its fusion protein) (e.g., in a dose-dependent manner) for binding to soluble hCD7.

[0217] Optionally, competition for binding to hCD7 is performed using SPR. SPR can be performed as described herein.

[0218] 30. The antibody or fragment according to any of the preceding clauses, which specifically binds to: human CD7 comprising SEQ ID NO: 82; and / or cynomolgus monkey CD7 comprising SEQ ID NO: 85; and / or rat CD7 comprising SEQ ID NO: 86.

[0219] In one example, the CD7 herein is human, mouse or cynomolgus monkey CD7.

[0220] In one embodiment, the antibody or fragment binds to cynomolgus monkey CD7 with an affinity of less than 1 nM (e.g., 1 nM to 0.01 pM, or 1 nM to 0.1 pM, or 1 nM to 1 pM). In one embodiment, the antibody or fragment binds to cynomolgus monkey CD7 with an affinity of less than 10 nM (e.g., 10 nM to 0.01 pM, or 10 nM to 0.1 pM, or 10 nM to 1 pM). In one embodiment, the antibody or fragment binds to cynomolgus monkey CD7 with an affinity of less than 0.1 nM (e.g., 0.1 nM to 0.01 pM, or 0.1 nM to 0.1 pM, or 0.1 nM to 1 pM). In one embodiment, the antibody or fragment binds to cynomolgus monkey CD7 with an affinity of less than 0.01 nM (e.g., 0.011 nM to 0.01 pM, or 0.01 nM to 0.1 pM).

[0221] In one embodiment, the antibody or fragment binds to cynomolgus monkey CD7 with an affinity within 2 times that of hCD7. In one embodiment, the antibody or fragment binds to cynomolgus monkey CD7 with an affinity within 4 times that of hCD7. In one embodiment, the antibody or fragment binds to cynomolgus monkey CD7 with an affinity within 5 times that of hCD7. In one embodiment, the antibody or fragment binds to cynomolgus monkey CD7 with an affinity within 6 times that of hCD7. In one embodiment, the antibody or fragment binds to cynomolgus monkey CD7 with an affinity within 8 times that of hCD7. In one embodiment, the antibody or fragment binds to cynomolgus monkey CD7 with an affinity within 10 times that of hCD7.

[0222] "hCD7" herein is human CD7, such as human CD7 disclosed herein.

[0223] In one embodiment, the antibody or fragment does not detectably bind to cynomolgus monkey CD7. In one embodiment, the antibody or fragment does not detectably bind to murine (e.g., mouse and / or rat) CD7.

[0224] In one embodiment, the antibody or fragment binds to murine (e.g., mouse and / or rat) CD7 with an affinity of less than 1 nM (e.g., 1 nM to 0.01 pM, or 1 nM to 0.1 pM, or 1 nM to 1 pM). In one embodiment, the antibody or fragment binds to murine CD7 with an affinity of less than 10 nM (e.g., 10 nM to 0.01 pM, or 10 nM to 0.1 pM, or 10 nM to 1 pM). In one embodiment, the antibody or fragment binds to murine CD7 with an affinity of less than 0.1 nM (e.g., 0.1 nM to 0.01 pM, or 0.1 nM to 0.1 pM, or 0.1 nM to 1 pM). In one embodiment, the antibody or fragment binds to murine CD7 with an affinity of less than 0.01 nM (e.g., 0.011 nM to 0.01 pM, or 0.01 nM to 0.1 pM).

[0225] Optionally, the antibody or fragment comprises an effector-enabled constant region, such as a human constant region, for example an IgG1 constant region. Optionally, the antibody or fragment comprises a murine (e.g., mouse and / or rat) constant region. Optionally, the antibody or fragment comprises any heavy chain constant region sequence described herein.

[0226] 31. The antibody or fragment according to any of the preceding clauses, wherein the antibody or fragment comprises a human constant region, such as an IgG1 constant region.

[0227] Optionally, the constant region is an IgG1 constant region, optionally the constant region comprises any IgG1 constant region amino acid sequence disclosed herein. Optionally, the constant region is an IgG2 constant region, optionally the constant region comprises any IgG1 constant region amino acid sequence disclosed herein. Optionally, the constant region is an IgG1 constant region, optionally the constant region comprises any IgG3 constant region amino acid sequence disclosed herein. Optionally, the constant region is an IgG1 constant region, optionally the constant region comprises any IgG4 constant region amino acid sequence disclosed herein.

[0228] In an example (optionally in addition to a heavy chain region according to the immediately above paragraph), the constant region comprises a light chain constant region comprising any of the light chain constant region amino acid sequences disclosed herein.

[0229] 32. An antibody or fragment according to clause 31, wherein the constant region is an IgG1 constant region, optionally the constant region comprises the amino acid sequence of SEQ ID NO: 88, 90, 92, 94 or 96 (eg, SEQ ID NO: 88).

[0230] In other embodiments, the antibody or fragment is any isotype or constant region as defined herein. In one embodiment, the constant region is wild-type human IgG1. For example, the constant region is an effector-enabled IgG1 constant region, optionally with ADCC and / or CDC activity. In one embodiment, the constant region is engineered for enhanced ADCC and / or CDC and / or ADCP. In another embodiment, the constant region is engineered for enhanced effector function.

[0231] The efficacy of Fc-mediated effects can be enhanced by engineering the Fc domain by any technique apparent to those skilled in the art. In another embodiment, the antibodies and fragments disclosed herein may include a triple mutation (M252Y / S254T / T256E) that enhances binding to FcRn.

[0232] 33. An antibody or fragment (eg, a bispecific antibody) according to any of the preceding clauses, further comprising an antigen binding site that specifically binds to another target antigen (optionally human CD5, CD14 or CD1g, eg, for treating leukemias such as AML).

[0233] For example, the another target antigen is human CD5.

[0234] In examples, the additional binding site is an agonist binding site for the other antigen. In examples, the additional binding site is an antagonist binding site for the other antigen.

[0235] In an example, the additional binding site is an antibody binding site comprising VH and VL; a binding site comprised by the constant domain of an antibody (e.g., an Fcab binding site) or a non-immunoglobulin binding site (e.g., a fibronectin domain). Optionally, the antigen binding site is any antigen binding site disclosed herein.

[0236] For example, the antibody or fragment is a bispecific antibody or fragment. For example, the antibody or fragment is a dual binding antibody or fragment, or a fusion protein comprising an antibody or fragment thereof according to any of the preceding clauses. Dual binding antibody has the meaning as described above.

[0237] In an example, the antibody, fragment or fusion protein comprises a bispecific format selected from DVD-Ig, mAb 2 , FIT-Ig, mAb-dAb, dock and lock, SEEDbody, scDiabody-Fc, diabody-Fc, tandem scFv-Fc, Fab-scFv-Fc, Fab-scFv, intrabody, BiTE, diabody, DART, TandAb, scDiabody, scDiabody-CH3, Diabody-CH3, minibody, knob-in-hole, knob-in-hole with common light chain, knob-in-hole with common light chain and charge pair, charge pair, charge pair with common light chain, specifically mAb 2 , protuberances into holes, protuberances into holes with common light chains, protuberances into holes with common light chains and charge pairs, and FIT-Ig, such as mAbs 2 and FIT-Ig.

[0238] In one embodiment, the bispecific format is selected from DVD-Ig, mAb 2, FIT-Ig, mAb-dAb, docking and locking, Fab-arm exchange, SEEDbody, triabody, LUZ-Y, Fcab, kλ body, orthogonal Fab, scDiabody-Fc, diabody Fc, tandem scFv-Fc, Fab-scFv-Fc, Fab-scFv, intrabody, BiTE, diabody, DART, TandAb, scDiabody, scDiabody-CH3, diabody-CH3, tripartite, minibody, minibody, TriBi minibody, scFv-CH3 KIH, scFv-CH-CL-scFv, F(ab′)2-scFv, scFv-KIH, Fab-scFv-Fc, tetravalent HCab, ImmTAC, knobs-in-holes, knobs-in-holes with common light chain, knobs-in-holes with common light chain and charge pair, charge pair, charge pair with common light chain, DT-IgG, DutaMab, IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig and zybody.

[0239] In one embodiment, the bispecific antibody is selected from DVD-Ig, FIT-Ig, mAb-dAb, docking and locking, Fab-arm exchange, SEEDbody, trifunctional antibody, LUZ-Y, Fcab, κλ body, orthogonal Fab, scDiabody-Fc, bifunctional antibody Fc, tandem scFv-Fc, Fab-scFv-Fc, Fab-scFv, intrabody, BiTE, bifunctional antibody, DART, TandAb, scDiabody, scDiabody-CH3, bifunctional antibody-CH3, triplet, minibody, minibody, TriBi minibody, scFv-CH3 KIH, scFv-CH-CL-scFv, F(ab′)2-scFv, scFv-KIH, Fab-scFv-Fc, tetravalent HCab, ImmTAC, knobs-in-holes, knobs-in-holes with common light chain, knobs-in-holes with common light chain and charge pair, charge pair, charge pair with common light chain, DT-IgG, DutaMab, IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig and zybody, such as DVD-Ig, FIT-Ig, mAb-dAb, dock and lock, SEEDbody, scDiabody-Fc, diabody-Fc, tandem scFv-Fc, Fab-scFv-Fc, Fab-scFv, intrabody, BiTE, diabody, DART, TandAb, scDiabody, scDiabody-CH3, Diabody-CH3, minibody, knobs-in-hole, knobs-in-hole with common light chain, knobs-in-hole with common light chain and charge pair, charge pair, charge pair with common light chain, specifically knobs-in-hole, knobs-in-hole with common light chain, knobs-in-hole with common light chain and charge pair, and FIT-Ig, such as FIT-Ig.

[0240] In one embodiment, the bispecific antibody is selected from DVD-Ig, mAb 2, mAb-dAb, docking and locking, Fab-arm exchange, SEEDbody, triabody, LUZ-Y, Fcab, κλ body, orthogonal Fab, scDiabody-Fc, diabody Fc, tandem scFv-Fc, Fab-scFv-Fc, Fab-scFv, intrabody, BiTE, diabody, DART, TandAb, scDiabody, scDiabody-CH3, diabody-CH3, tripartite, minibody, minibody, TriBi minibody, scFv-CH3 KIH, scFv-CH-CL-scFv, F(ab′)2-scFv, scFv-KIH, Fab-scFv-Fc, tetravalent HCab, ImmTAC, knobs-in-holes, knobs-in-holes with common light chain, knobs-in-holes with common light chain and charge pairs, charge pairs, charge pairs with common light chain, DT-IgG, DutaMab, IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig and zybody, such as DVD-Ig, mAb 2 , mAb-dAb, dock and lock, SEEDbody, scDiabody-Fc, diabody-Fc, tandem scFv-Fc, Fab-scFv-Fc, Fab-scFv, intrabody, BiTE, diabody, DART, TandAb, scDiabody, scDiabody-CH3, Diabody-CH3, minibody, knob-in-hole, knob-in-hole with common light chain, knob-in-hole with common light chain and charge pair, charge pair, charge pair with common light chain, specifically mAb 2 , protuberances into holes, protuberances into holes with common light chains and charge pairs, and protuberances into holes with common light chains, such as mAbs 2 .

[0241] In one embodiment, the bispecific antibody is selected from DVD-Ig, mAb-dAb, docking and locking, Fab-arm exchange, SEEDbody, trifunctional antibody, LUZ-Y, Fcab, κλ body, orthogonal Fab, scDiabody-Fc, bifunctional antibody Fc, tandem scFv-Fc, Fab-scFv-Fc, Fab-scFv, intrabody, BiTE, bifunctional antibody, DART, TandAb, scDiabody, scDiabody-CH3, bifunctional antibody-CH3, triplet, minibody, minibody, TriBi minibody, scFv-CH3 KIH, scFv-CH-CL-scFv, F(ab′)2-scFv, scFv-KIH, Fab-scFv-Fc, tetravalent HCab, ImmTAC, knobs-in-holes, knobs-in-holes with common light chain, knobs-in-holes with common light chain and charge pair, charge pair, charge pair with common light chain, DT-IgG, DutaMab, IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig and zybody, such as DVD-Ig, mAb-dAb, dock and lock, SEEDbody, scDiabody-Fc, diabody-Fc, tandem scFv-Fc, Fab-scFv-Fc, Fab-scFv, intrabody, BiTE, diabody, DART, TandAb, scDiabody, scDiabody-CH3, Diabody-CH3, minibody, knobs-in-holes, knobs-in-holes with common light chain, knobs-in-holes with common light chain and charge pairs, charge pairs, charge pairs with common light chain, specifically knobs-in-holes, knobs-in-holes with common light chain and charge pairs, and knobs-in-holes with common light chain.

[0242] 34. An anti-CD7 antibody or fragment as defined in any one of the preceding clauses for use in treating or preventing a CD7-mediated disease or condition (optionally cancer, such as leukemia or lymphoma) in a subject.

[0243] 35. An anti-CD7 antibody or fragment according to clause 34, wherein the disease or condition is selected from leukemia, lymphoma, blood cancer and myelodysplastic syndrome (MDS).

[0244] In an example, the subject is a human. In an alternative, the subject is a non-human animal. In an example, the subject is an adult. In an example, the subject is a pediatric human.

[0245] In an example, the antibodies or fragments described herein are used to treat or prevent a disease or condition in a subject (eg, a human) selected from

[0246] T-cell acute lymphoblastic leukemia;

[0247] Acute myeloid leukemia with CD7 expression;

[0248] Precursor T-cell lymphoblastic lymphoma;

[0249] T-cell prolymphocytic leukemia;

[0250] T-cell large granular lymphocytic leukemia;

[0251] Peripheral T-cell lymphoma;

[0252] Angioimmunoblastic T-cell lymphoma;

[0253] Extranodal NK / T-cell lymphoma, nasal type;

[0254] Enteropathic intestinal T-cell lymphoma; and

[0255] Hepatosplenic T-cell lymphoma.

[0256] In examples, the disease or condition is in a human. In examples, the disease or condition is in an animal.

[0257] In an example, the antibody or fragment of the invention is used to treat or prevent a CD7-mediated disease or condition in a human, the disease or condition being selected, for example, from a tumor or non-tumor disease, a chronic viral infection and a malignancy such as melanoma, Merkel cell carcinoma, non-small cell lung cancer (squamous and non-squamous), renal cell carcinoma, bladder cancer, head and neck squamous cell carcinoma, mesothelioma, virus-induced cancers (such as cervical cancer and nasopharyngeal carcinoma), soft tissue sarcoma, hematological malignancies such as Hodgkin's disease and non-Hodgkin's disease and diffuse large B-cell lymphoma.

[0258] In an example, the CD7-mediated disease or condition is a neurodegenerative disease, disorder or condition, such as selected from Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, primary progressive multiple sclerosis, secondary progressive multiple sclerosis, corticobasal degeneration, Rett syndrome, a retinal degeneration disorder selected from age-related macular degeneration and retinitis pigmentosa; anterior ischemic optic neuropathy, glaucoma, uveitis, depression, trauma-related stress or post-traumatic stress disorder, frontotemporal dementia, Lewy body dementia, mild cognitive impairment, posterior cortical atrophy, primary progressive aphasia and progressive supranuclear palsy or senile dementia, specifically, the neurodegenerative disease, disorder or condition is selected from Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease and Huntington's disease, such as Alzheimer's disease.

[0259] In an example, the antibody, fragment, combination of the present invention is administered intravenously to the subject; or is used for intravenous administration to the subject. In an example, the antibody, fragment, combination of the present invention is administered subcutaneously to the subject; or is used for subcutaneous administration to the subject.

[0260] 36. The antibody or fragment according to clause 33, wherein the antibody or fragment is administered to the subject simultaneously or sequentially with chemotherapy, radiotherapy or an immune checkpoint inhibitor.

[0261] Optionally, the chemotherapy is selected from nelarabine; cyclophosphamide; vincristine; doxorubicin; and dexamethasone alternating with methotrexate and cytarabine.

[0262] 37. A combination of an amount of an anti-CD7 antibody or fragment and an amount of a chemotherapeutic agent (optionally comprising multiple doses of said antibody and / or agent), wherein said antibody or fragment is an antibody or fragment according to any one of clauses 1 to 36.

[0263] Also provided is a medical kit comprising the combination, a first sterile container comprising the amount of the antibody or fragment and a second sterile container comprising the amount of the chemotherapeutic agent, and optionally instructions for using the combination to treat cancer in a subject.

[0264] 38. The antibody, fragment or combination according to any one of clauses 1 to 37 for use in a method of treating leukemia in a human, wherein the antibody, fragment or combination is optionally administered to the human together with an antagonist of human CD5, CD14 or CD19.

[0265] 39. The antibody, fragment or combination according to clause 38, wherein the leukemia is acute myeloid leukemia or T-ALL, or is a relapsed leukemia (eg, relapsed AML or T-ALL).

[0266] 40. The antibody, fragment or combination according to any one of clauses 1 to 37 for use in treating a human or animal subject suffering from a condition caused by CD7 + A method for treating a cell-mediated disease or condition, wherein the method comprises administering to the subject the antibody, combined fragment, wherein CD7 cells are targeted and killed, optionally by ADCP and / or CDC.

[0267] 41. Use of an antibody, fragment or combination according to any preceding clause in the manufacture of a medicament for administration to a subject to treat or prevent a CD7-mediated disease or condition, optionally cancer.

[0268] 42. A method of treating or preventing a CD7-mediated disease or condition (optionally cancer) in a subject, the method comprising administering to the subject a therapeutically effective amount of an antibody, fragment or combination according to any one of clauses 1 to 40, wherein the CD7-mediated disease or condition is thereby treated or prevented.

[0269] 43. The use according to clause 41 or the method according to clause 42, wherein the CD7-mediated disease or condition is leukemia (optionally T-ALL).

[0270] 44. The antibody, fragment, combination, use or method according to any one of clauses 34 to 43, further comprising administering to the subject an additional therapy, such as an additional therapeutic agent, optionally wherein the additional therapeutic agent is selected from the group consisting of:

[0271] a. Cyclophosphamide;

[0272] b. Vincristine;

[0273] c. Doxorubicin;

[0274] d. Dexamethasone;

[0275] e.Methotrexate;

[0276] f. cytarabine; and

[0277] g. Nelarabine.

[0278] 45. A pharmaceutical composition comprising an antibody, fragment or combination according to any one of clauses 1 to 40 and 44 and a pharmaceutically acceptable excipient, diluent or carrier, and optionally in combination with an additional therapeutic agent selected from the agents described in clause 44.

[0279] 46. ​​A pharmaceutical composition according to clause 45 for use in the treatment and / or prevention of a CD7-mediated condition or disease, optionally cancer.

[0280] 47. A pharmaceutical composition according to clause 45 or 46, in combination with a label or instructions for use in treating and / or preventing said disease or condition in a human; optionally, wherein said label or instructions comprises a marketing authorization number (optionally, an FDA or EMA authorization number); optionally wherein said kit comprises an IV or injection device comprising said antibody or fragment.

[0281] 48. A nucleic acid encoding the VH domain and / or VL domain of the antibody or fragment according to any one of clauses 1 to 33.

[0282] 49. A nucleic acid encoding: a VH domain comprising the amino acid sequence of a VH domain of an antibody selected from the group consisting of G09, F05, C02, and E04; or amino acids at least 70, 80, 85, 90, 95, 96, 97, 98, or 99% identical thereto.

[0283] For example, the identity is at least 85%. For example, the identity is at least 90%. For example, the identity is at least 95%.

[0284] 50. A nucleic acid encoding: a VL domain comprising the amino acid sequence of a VL domain of an antibody selected from the group consisting of G09, F05, C02, and E04; or amino acids at least 70, 80, 85, 90, 95, 96, 97, 98, or 99% identical thereto.

[0285] Optionally, the nucleic acid also encodes a VH domain comprising the amino acid sequence of the VH domain of the selected antibody; or an amino acid that is at least 70, 80, 85, 90, 95, 96, 97, 98 or 99% identical thereto. For example, the identity is at least 85%. For example, the identity is at least 90%. For example, the identity is at least 95%.

[0286] 51. A nucleic acid comprising

[0287] (a) a nucleotide sequence that is at least 70% identical to the sequence of SEQ ID NO: 10; and / or

[0288] (b) a nucleotide sequence that is at least 70% identical to the sequence of SEQ ID NO:20.

[0289] 52. A nucleic acid encoding the heavy and / or light chain of the antibody or fragment according to any one of clauses 1 to 33.

[0290] 53. A nucleic acid encoding a heavy chain comprising an amino acid sequence at least 70% identical to SEQ ID NO:8.

[0291] 54. A nucleic acid encoding a light chain comprising an amino acid sequence at least 70% identical to SEQ ID NO: 18.

[0292] 55. A nucleic acid (e.g., in a host cell, e.g., a CHO or HEK293 or Cos cell) comprising

[0293] (a) a nucleotide sequence that is at least 70% identical to a selected heavy chain sequence of an antibody selected from G09, F05, C02 and E04; and / or

[0294] (b) a nucleotide sequence that is at least 70% identical to a selected sequence of an antibody selected from the group consisting of G09, F05, C02, and E04.

[0295] In any instance herein where % identity is mentioned, there is 100% identity in that instance.

[0296] 56. A nucleic acid encoding the heavy and / or light chain of the antibody or fragment according to any one of clauses 1 to 32.

[0297] All nucleic acids of the invention can be expressed in host cells such as CHO or HEK293 or Cos cells, for example for the expression of variable domains or chains of antibodies or fragments of the invention.

[0298] 57. A vector comprising the nucleic acid (eg, a nucleic acid according to any one of clauses 48 to 55); optionally wherein the vector is a CHO or HEK293 vector.

[0299] 58. A host cell comprising the nucleic acid (eg, a nucleic acid according to any one of clauses 48 to 55) or the vector according to clause 57.

[0300] 59. An antibody, fragment, combination, vector, host cell, composition, use or method according to any of the preceding clauses, wherein the antibody or fragment comprises an IgG1 (eg, human IgG1 or IgG1*01) constant region.

[0301] 60. The antibody, fragment, combination, vector, host cell, composition, use or method of clause 59, wherein the constant region comprises a glycine at position 430, an arginine at position 356 and / or an arginine at position 357 in the IgG1 CH3 region (according to EU numbering).

[0302] 61. The antibody, fragment, combination, vector, host cell, composition, use or method of clause 59, wherein the constant region comprises a glycine at position 430.

[0303] 62. An antibody, fragment, combination, vector, host cell, composition, use or method as described herein.

[0304] The present invention provides:

[0305] A method of diagnosing a CD7-mediated disease or condition (optionally cancer) in a subject, the method comprising combining an antibody or fragment of the invention with an isolated cell sample (e.g., a blood or serum sample) and determining that cells comprised by the sample are specifically bound by the antibody or fragment.

[0306] Also provided:

[0307] An in vitro assay for detecting CD7-positive cells in a sample, the assay comprising combining an antibody or fragment of the invention with an isolated cell sample (eg, a blood or serum sample) and determining that cells comprised by the sample are specifically bound by the antibody or fragment.

[0308] The disease or condition may be any disease or condition disclosed herein. Detection may be by any conventional means, for example, using a label such as a fluorescent label, ELISA or RIA.

[0309] In an example, the antibody or fragment comprises a HCDR3 length of 9, 10, 11 or 12 residues, such as 10, such as 11 residues. In an example, the antibody or fragment comprises a LCDR3 length of 7, 8 or 9 residues, such as 8, such as 9 residues. In an example, each VH domain of the antibody or fragment comprises 1-11 non-germline residues, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 non-germline residues. In an example, each VL domain of the antibody or fragment comprises 3-8 non-germline residues, such as 3, 4, 5, 6, 7 or 8 non-germline residues.

[0310] In an embodiment, the CDR sequences herein are determined according to Kabat. In an alternative, the CDR sequences are determined according to IMGT.

[0311] In examples, the selected antibody is G09.

[0312] In an example, the selected antibody comprises a heavy chain of G09, F05, C02 or E04. In an example, the selected antibody comprises a heavy chain of G09.

[0313] In an example, the heavy chain of the antibody or fragment of the present invention is a human γ-1, γ-2, γ-3, γ-4, μ, δ, ε or α isotype, preferably a γ isotype (e.g., an IgG4 isotype). In an example, the light chain of the antibody or fragment of the present invention includes a human κ constant region. Alternatively, in an example, the light chain of the antibody or fragment of the present invention includes a human λ constant region.

[0314] Optionally, the antibody is a 4-chain antibody comprising a heavy chain dimer associated with a dimer of a light chain. In an example, the heavy chain comprises one or heavy chain CDR or CDR combination as disclosed herein and / or the light chain comprises one or heavy chain CDR or CDR combination as disclosed herein, such as from the same selected antibody. In an example, the heavy chain comprises a VH domain as disclosed herein and / or the light chain comprises a VL as disclosed herein, such as from the same selected antibody. In an example, the heavy chain and the light chain are from the same selected antibody, such as any antibody disclosed in the sequence listing herein or in the table in the examples herein.

[0315] In an example, the selected antibody includes a light chain of G09, F05, C02 or E04. In an example, the selected antibody includes a light chain of G09.

[0316] In examples, the selected antibody comprises the variable domain of G09, F05, C02 or E04. In examples, the selected antibody comprises the variable domain of G09.

[0317] In an example, the selected antibody comprises the VH domain of G09, F05, C02 or E04. In an example, the selected antibody comprises the VH domain of G09.

[0318] In an example, the selected antibody includes the VH domain and the VL domain of G09, F05, C02 or E04. In an example, the selected antibody includes the VH domain and the VL domain of G09.

[0319] In an example, the binding site of the antibody or fragment comprises a VH / VL pair that specifically binds to human CD7.

[0320] Optionally, the antibody or fragment competes with G09 (eg, G09 in IgG format, eg, human IgG1 ) for binding to CD7 as determined by SPR.

[0321] Optionally, the amino acid substitutions are conservative amino acid substitutions, optionally wherein each conservative substitution is from group (1) to (6):

[0322] 1) Alanine (A), serine (S), threonine (T);

[0323] 2) Aspartic acid (D), glutamic acid (E);

[0324] 3) Asparagine (N), glutamine (Q);

[0325] 4) Arginine (R), Lysine (K);

[0326] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

[0327] 6) Phenylalanine (F), tyrosine (Y), tryptophan (W).

[0328] Any SPR herein is, for example, surface plasmon resonance (SPR) at 37°C and pH 7.6.

[0329] Optionally, any CD7 herein is (eg, in an in vitro assay) human CD7, eg, comprising the amino acid sequence of human CD7 disclosed herein.

[0330] In an example, the antibody or fragment of the invention is expressed in an amount of, for example, 5×10 6 M -1 ×s -1 ; or about 5×10 6 M -1 ×s -1 In an example, the antibody or fragment of the invention binds to human CD7 with a Ka of 4 or 5 s -1 ; or about 4 or 5s -1 In an example, the antibody or fragment of the invention binds to human CD7 with a KD of, for example, 0.07 or 0.14 nM; or about 0.07 or 0.14 nM. In an embodiment, the fragment is a Fab fragment. In an embodiment, the fragment is a scFv.

[0331] Optionally, said antibody of the invention binds to CD7 with an affinity (KD) of 1 pM to 5 nM, optionally wherein binding is determined by SPR at 37°C, at pH 7.6, using a Fab of said antibody.

[0332] Optionally, the antibody has a dissociation rate (K) of binding to CD7. off ) is 1×10 -5 S -1 to 1×10 -3 S -1 , optionally wherein binding is determined by SPR at 37°C at pH 7.6 using a Fab of said antibody.

[0333] Optionally, the binding rate (K) of the antibody to CD7 on ) is 1×10 5 M -1 S -1to 1×10 7 M -1 S -1 , optionally wherein binding is determined by SPR at 37°C at pH 7.6 using a Fab of said antibody.

[0334] In an example, the antibody (e.g., as a Fab) or fragment binds CD7 (e.g., human CD7) with an affinity (KD) of

[0335] (a) 2 pM, 3 pM, 4 pM, 5 pM or 10 pM to 3 nM, 4 nM or 5 nM;

[0336] (b) 1 pM to 10 pM to 5 nM;

[0337] (c) 10 pM to 3 nM, 4 nM or 5 nM;

[0338] (d) 50 pM or 80 pM to 200 nM;

[0339] (e) 50 pM or 80 pM to 150 nM; or

[0340] (f) 50 pM or 80 pM to 100 nM.

[0341] In an example, the KD is (or is about) 5 pM to 15 pM (e.g., 10 pM). In an example, the KD is (or is about) 2 nM to 5 nM (e.g., 3 nM). In an example, the KD is (or is about) 100 pM to 400 pM (e.g., 140 pM or 390 pM).

[0342] In an example, the off-rate (K) of an antibody (e.g., as a Fab) or fragment binding to CD7 (e.g., human CD7) is off )for

[0343] (a)1×10 -5 S -1 to 5×10 -4 S -1 ;

[0344] (b)1×10 -5 S -1 to 6×10 -4 S -1 ;

[0345] (c)1×10 -5 S -1 to 7×10 -4 S -1 ;

[0346] (d)1×10 -5 S -1 to 8×10-4 S -1 ;

[0347] (e)2×10 -5 S -1 to 1×10 -3 S -1 ;

[0348] (f)2×10 -5 S -1 to 5×10 -4 S -1 ;

[0349] (g)2×10 -5 S -1 to 6×10 -4 S -1 ;

[0350] (h)2×10 -5 S -1 to 7×10 -4 S -1 ;or

[0351] (i)2×10 -5 S -1 to 8×10 -4 S -1 .

[0352] In this example, K off is (or is approximately) 5×10 -4 S -1 (For example, when KD is (or is approximately) 2nM to 400pM; when KD is (or is approximately) 2nM to 5nM (e.g., 3nM); or when KD is (or is approximately) 100pM to 400pM (e.g., 140pM or 390pM). In an example, K off is (or is approximately) 3×10 -5 S -1 (For example, when KD is (or is about) 5 pM to 15 pM (eg, 10 pM)).

[0353] In an example, the binding rate (K) of the antibody (e.g., as a Fab) or fragment to CD7 (e.g., human CD7) is on )for

[0354] (a)1×10 5 M -1 S -1 to 1×10 6 M -1 S -1 ;

[0355] (b)1×105 M -1 S -1 to 2×10 6 M -1 S -1 ;

[0356] (c)1×10 5 M -1 S -1 to 3×10 6 M -1 S -1 ;

[0357] (d)1×10 5 M -1 S -1 to 4×10 6 M -1 S -1 ;

[0358] (e)1×10 5 M -1 S -1 to 5×10 6 M -1 S -1 ;

[0359] (f)2×10 5 M -1 S -1 to 5×10 6 M -1 S -1 ;

[0360] (g)3×10 5 M -1 S -1 to 5×10 6 M -1 S -1 ;

[0361] (h)4×10 5 M -1 S -1 to 5×10 6 M -1 S -1 ;

[0362] (i)5×10 5 M -1 S -1 to 5×10 6 M -1 S -1 ;or

[0363] (i)6×10 5 M -1 S-1 to 5×10 6 M -1 S -1 .

[0364] In this example, K on is (or is approximately) 1×10 -5 M -1 S -1 or 2×10 -5 M -1 S -1 (For example, when KD is 2nM to 5nM (eg, 3nM)). In an example, K on is (or is approximately) 1×10 -6 M -1 S -1 to 4×10 -6 M -1 S -1 , 1×10 -6 M -1 S -1 , 2×10 -6 M -1 S -1 , 3×10 -6 M -1 S -1 or 4×10 -6 M -1 S -1 (For example, when KD is (or is about) 5 pM to 400 pM (eg, 140 pM or 390 pM) or 5 pM to 15 pM (eg, 10 pM)).

[0365] As provided in the clauses or other aspects herein, the anti-CD7 antibody or fragment can be prepared with a K of less than 50 nM, less than 40 nM, less than 30 nM. D Binds to CD7, such as human CD7, as determined by surface plasmon resonance. In another embodiment, the anti-CD7 antibody or fragment can bind to CD7 with a K of less than 20 nM, less than 15 nM, less than 10 nM, or less than 10 nM. D Binds to CD7, e.g., human CD7, as determined by surface plasmon resonance. The anti-CD7 antibody or fragment may bind to CD7 with a K of less than 8 nM, less than 5 nM, less than 4 nM, less than 3 nM, less than 2 nM, or less than 1 nM. D Binds to CD7, e.g., human CD7, as determined by surface plasmon resonance. K D It may be 0.9 nM or less, 0.8 nM or less, 0.7 nM or less, 0.6 nM or less, 0.5 nM or less, 0.4 nM or less, 0.3 nM or less, 0.2 nM or less, or 0.1 nM or less.

[0366] In another embodiment, K D In the range of 0.01 to 1 nM, or in the range of 0.05 to 2 nM, or in the range of 0.05 to 1 nM. K D It may be associated with hCD7, cynomolgus monkey (ie, "cynomolgus") CD7 and / or mouse CD7.

[0367] In another embodiment, the anti-CD7 antibodies described herein have a K of about 0.5 to 10 μM, such as about 1 to 8 μM or about 1 to 7 μM. ON In another embodiment, K ON The rate is about 1 to 5 μM, such as about 1 μM, about 1.5 μM, about 2 μM, about 2.5 μM, or about 3 μM. In another embodiment, K ON The rate is about 3.5 μM, about 4 μM, about 4.5 μM, about 5 μM, or about 5.5 μM.

[0368] In another embodiment, the anti-CD7 antibodies described herein have a K of about 0.01 to 100 mM, such as about 0.1 to 50 mM or about 0.5 to 50 mM. OFF In another embodiment, K OFF The rate is about 0.5 to 10 mM or about 0.5 to 10 mM, such as about 1 mM, about 2 mM, about 3 mM, about 4 mM or about 5 mM. In another embodiment, K OFF The rate is about 0.6 mM, about 0.7 mM, about 0.8 mM, or about 0.9 mM.

[0369] In an example, the antibody or fragment of the invention comprises the VH domain and VL domain of G09, F05, C02 and E04.

[0370] In an example, the antibody or fragment of the present invention comprises the VH domain and VL domain of G09. In an example, the antibody or fragment of the present invention comprises the VH domain and VL domain of F05. In an example, the antibody or fragment of the present invention comprises the VH domain and VL domain of G09. In an example, the antibody or fragment of the present invention comprises the VH domain and VL domain of C02. In an example, the antibody or fragment of the present invention comprises the VH domain and VL domain of E04.

[0371] In examples, the selected antibody is G09.

[0372] In an example, the selected antibody comprises the variable domain of an antibody selected from G09, F05, C02 and E04.

[0373] In an example, the selected antibody comprises a VH domain of an antibody selected from G09, F05, C02 and E04. In an example, the selected antibody comprises a VH domain of G09.

[0374] In an example, the selected antibody comprises a VH domain and a VL domain of an antibody selected from G09, F05, C02 and E04. In an example, the selected antibody comprises a VH domain and a VL domain of G09.

[0375] Optionally, the antibody or fragment of the invention comprises HCDR3 of an antibody selected from G09, F05, C02 and E04. Optionally, the antibody or fragment of the invention comprises HCDR1 and / or HCDR2 of said antibody.

[0376] Optionally, the antibody or fragment of the invention comprises HCDR1 of an antibody selected from G09, F05, C02 and E04. Optionally, the antibody or fragment of the invention comprises HCDR2 and / or HCDR3 of said selected antibody.

[0377] Optionally, the antibody or fragment of the invention comprises HCDR2 of an antibody selected from G09, F05, C02 and E04. Optionally, the antibody or fragment of the invention comprises HCDR1 and / or HCDR3 of said antibody.

[0378] Optionally, the antibody or fragment of the invention comprises the VH of an antibody selected from G09, F05, C02 and E04. Optionally, the antibody or fragment of the invention comprises the VL of said selected antibody.

[0379] Optionally, the antibody or fragment of the invention comprises the VL of an antibody selected from G09, F05, C02 and E04. Optionally, the antibody or fragment of the invention comprises the VH of said selected antibody.

[0380] Optionally, the antibody or fragment of the invention comprises a heavy chain of an antibody selected from G09, F05, C02 and E04. Optionally, the antibody or fragment of the invention comprises a light chain of said selected antibody.

[0381] Optionally, the antibody or fragment of the invention comprises a light chain of an antibody selected from G09, F05, C02 and E04. Optionally, the antibody or fragment of the invention comprises a heavy chain of the selected antibody. In an example, the selected antibody is G09.

[0382] Optionally, the antibodies of the invention include a human IgG1*01 constant region. Optionally, the antibodies of the invention include a human IgG1E430G constant region, such as an IgG1*01 E430G constant region. Optionally, the antibodies of the invention include a human IgG1 E345R constant region, such as an IgG1*01 E345R constant region.

[0383] Preferably, an antibody or fragment thereof that specifically binds to hCD7 does not cross-react with other antigens (but may optionally cross-react with different CD7 species such as rhesus monkey, cynomolgus monkey or mouse). For example, the antibody or fragment thereof may be cross-reacted with other antigens (but may optionally cross-react with different CD7 species such as rhesus monkey, cynomolgus monkey or mouse). TM Or other techniques known to those skilled in the art to identify antibodies or fragments thereof that specifically bind to the CD7 antigen. As determined using experimental techniques such as radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA), when the affinity of the antibody or its fragment to the hCD7 antigen is higher than the affinity to any cross-reactive antigen, the antibody or its fragment specifically binds to the hCD7 antigen. Typically, a specific or selective reaction will be at least twice the background signal or noise, and more typically more than 10 times the background. For a discussion of antibody specificity, see, for example, Paul, ed., 1989, Basic Immunology, 2nd edition, Raven Press, New York, pp. 332-336.

[0384] The contact amino acid residues involved in the interaction between an antibody and an antigen such as CD7 can be determined by various methods known to those skilled in the art.

[0385] In one embodiment, if the antibody recognizes a linear epitope, short peptides based on the antigen sequence can be generated and binding of the antibody to these peptides can be assessed using standard techniques.

[0386] In one embodiment, limited proteolytic digestion and mass spectrometry can be used to identify binding epitopes.

[0387] In one embodiment, the contact residues of the epitope are identified by X-ray crystallography. In one embodiment, the contact residues of the epitope are identified by cryo-electron microscopy. In one embodiment, the contact residues of the epitope are identified by a combination of limited proteolytic digestion and mass spectrometry.

[0388] In another embodiment, the anti-CD7 antibodies (and fragments) described herein provide improved transient expression levels than other anti-CD7 antibodies and fragments. Thus, in one embodiment, the anti-CD7 antibodies (or fragments) are expressed in HEK293 cells, such as HEK293T cells, at an expression level of about 100 μg / mL or in a range of about 100 to 350 μg / mL. In another embodiment, the expression level is greater than about 350 μg / mL.

[0389] In another embodiment, the anti-CD7 antibody (or fragment) is expressed in CHO cells, such as Expi-CHO cells, at an expression level of about 100 μg / mL or in the range of about 100 to 350 μg / mL. In another embodiment, the expression level is greater than about 350 μg / mL.

[0390] In another embodiment, the anti-CD7 antibody (or fragment) is expressed at an expression level of about 100 μg / mL or in a range of about 100 to 350 μg / mL in CHO cells, such as Expi-CHO cells or CHO-E7EBNA cells. In another embodiment, the expression level is greater than about 350 μg / mL. For example, the antibody includes the VH domain and VL domain of any one of G09, and the format is human IgG1 or human IgG4 (e.g., IgG4-PE).

[0391] In any of these expression systems, expression is carried out in a range of about 0.5 mL and 3 mL, for example, about 0.5 mL and 2 mL. In any of these expression systems, anti-CD7 antibodies (or fragments) can be expressed from pTT5 vectors. In any of these expression systems, anti-CD7 antibodies (or fragments) can be expressed in conjunction with lipid transfection reagents, and can optionally be expressed in CHO cells such as Expi-CHO cells. In any of these expression systems, anti-CD7 antibodies (or fragments) can be expressed in conjunction with PEI transfection reagents, and can optionally be expressed in CHO cells such as CHO-E7 EBNA cells. In any of these expression systems, anti-CD7 antibodies (or fragments) can be expressed in conjunction with helper plasmids (such as AKT helper plasmids), and can optionally be expressed in CHO cells such as CHO-E7 EBNA cells.

[0392] In any of these expression systems, the expression level is between about 100 μg / mL and about 1500 μg / mL, for example, between about 100 μg / mL and about 1000 μg / mL, or between about 200 μg / mL and about 1000 μg / mL, or between about 350 μg / mL and about 1000 μg / mL. In any of these expression systems, the lower limit of expression can be about 100 μg / mL, about 200 μg / mL, about 300 μg / mL or about 400 μg / mL. In another embodiment, the lower limit of expression can be about 500 μg / mL, about 600 μg / mL, about 700 μg / mL or about 800 μg / mL. In any of these expression systems, the upper limit of expression can be about 2000 μg / mL, about 1800 μg / mL, about 1600 μg / mL or about 1500 μg / mL. In another embodiment, the upper expression limit can be about 1250 μg / mL, about 1000 μg / mL, about 900 μg / mL, or about 800 μg / mL.

[0393] In another embodiment, the expression system is a Lonza expression system, e.g. System. In the Lonza expression system, it can be expressed in a range of about 30mL to 2L, for example, 50mL to 1L or 1L to 2L. In the Lonza expression system, the anti-CD7 antibody (or fragment) can be expressed in combination with electroporation, and optionally without any auxiliary plasmid. In the Lonza expression system, the anti-CD7 antibody (or fragment) can be expressed at a level of about 1g / L, or about 900mg / L, or about 800mg / L, or about 700mg / L. In another embodiment, the anti-CD7 antibody (or fragment) can be expressed at a level of about 600mg / L, or about 500mg / L, or about 400mg / L in the Lonza expression system. In the Lonza expression system, the anti-CD7 antibody (or fragment) can be expressed at a level between about 400mg / L and about 2g / L, for example, between about 500mg / L and about 1.5g / L or between about 500mg / L and about 1g / L. In another embodiment, the expression level is higher than 1g / L. In another embodiment, the anti-CD7 antibody provides improved half-life compared to other anti-CD7 antibodies.

[0394] In one embodiment, the antibody or fragment is a human antibody or fragment. In one embodiment, the antibody or fragment is a fully human antibody or fragment. In one embodiment, the antibody or fragment is a fully human monoclonal antibody or fragment.

[0395] In one embodiment, the antibody or fragment is a humanized antibody or fragment. In one embodiment, the antibody or fragment is a humanized monoclonal antibody or fragment.

[0396] Contact amino acid residues involved in the interaction between antibody and antigen can be determined by various methods known to those skilled in the art, such as alanine scanning, protein crystallography, mass spectrometry or any other technique obvious to those skilled in the art.

[0397] In one embodiment, the CDR includes one amino acid substitution, and the amino acid substitution may be a conservative amino acid substitution. In one embodiment, the CDR includes two amino acid substitutions, and the amino acid substitution may be a conservative amino acid substitution. In one embodiment, the CDR includes three amino acid substitutions, and the amino acid substitution may be a conservative amino acid substitution. In one embodiment, the CDR includes four amino acid substitutions, and the amino acid substitution may be a conservative amino acid substitution. In one embodiment, the CDR includes five amino acid substitutions, and the amino acid substitution may be a conservative amino acid substitution. In one embodiment, the CDR includes six amino acid substitutions, and the amino acid substitution may be a conservative amino acid substitution.

[0398] Amino acid substitutions include changes in which an amino acid is substituted with a different naturally occurring amino acid residue. Such substitutions can be classified as "conservative", in which the amino acid residue contained in the polypeptide is replaced with another naturally occurring amino acid having similar properties in terms of polarity, side chain function or size. Such conservative substitutions are well known in the art. Substitutions encompassed by the present invention can also be "non-conservative", in which the amino acid residues present in the peptide are replaced with amino acids having different properties, such as naturally occurring amino acids from different groups (e.g., replacing charged or hydrophobic amino acids; replacing acids with alanine), or alternatively, in which naturally occurring amino acids are replaced with unconventional amino acids.

[0399] In one embodiment, conservative amino acid substitutions are as described herein. For example, Y is substituted with F, T is substituted with S or K, P is substituted with A, E is substituted with D or Q, N is substituted with D or G, R is substituted with K, G is substituted with N or A, T is substituted with S or K, D is substituted with N or E, I is substituted with L or V, F is substituted with Y, S is substituted with T or A, R is substituted with K, G is substituted with N or A, K is substituted with R, A is substituted with S, K or P. In another embodiment, conservative amino acid substitutions may be where Y is substituted with F, T is substituted with A or S, I is substituted with L or V, W is substituted with Y, M is substituted with L, N is substituted with D, G is substituted with A, T is substituted with A or S, D is substituted with N, I is substituted with L or V, F is substituted with Y or L, S is substituted with A or T, and A is substituted with S, G, T or V.

[0400] aspect

[0401] Any of the following aspects can be combined with any of the features disclosed herein (e.g., with any of the embodiments claimed herein or any of the clauses herein). For example, the ligand in any of these aspects can be an antibody or fragment of the invention.

[0402] 1. An anti-CD7 ligand administered to a human patient for use in the patient's CD7 + The invention relates to a method for treating cancer by complement dependent cytotoxicity (CDC) of cells (eg, cancer cells), wherein the ligand comprises an antibody Fc region and a binding site for specifically binding to human CD7.

[0403] In examples, the CD7 is human CD7 and the patient is human.

[0404] In alternatives of any aspect, instead of cancer, the ligands herein are used to treat a disease or condition mediated by CD7+ cells, such as CD7+ T cells or NK cells. For example, the disease or condition is an autoimmune disease or condition. For example, the disease is graft versus host disease (GvHD). For example, the disease or condition is an inflammatory disease or condition.

[0405] Optionally, in an alternative of any aspect, the ligand is administered to the subject prophylactically to reduce the risk of developing cancer or a disease or condition.

[0406] Optionally, in any aspect, the cell is a cell of the patient's immune system. Optionally, in any aspect, the cell is a T cell and / or a NK cell. Optionally, in any aspect, the cell is a cell of a tissue, cell or organ transplant comprised by a human.

[0407] 2. An anti-CD7 ligand administered to a human patient for use in the patient's CD7 + Ligand-dependent phagocytosis of cells (eg, cancer cells) for treating cancer, the ligand comprising an antibody Fc region and a binding site for specific binding to human CD7.

[0408] 3. wherein the phagocytosis is antibody-dependent cellular phagocytosis (ADCP), wherein the antibody is the ligand.

[0409] In vivo, ADCP can be mediated by monocytes, macrophages, neutrophils, and dendritic cells through FcγRIIa, FcγRI, and FcγRIIIa. Although all three receptors can participate in ADCP, FcγRIIa is considered to be the primary Fc7 receptor involved in this process. In an example, ADCP includes macrophages and / or monocytes included in the patient to CD7+ Phagocytosis of cancer cells.

[0410] 4. An anti-CD7 ligand administered to a human patient for use in the patient's CD7 + Ligand-dependent cell-mediated cytotoxicity of cancer cells for treating cancer, wherein the ligand comprises an antibody Fc region and a binding site for specific binding to human CD7.

[0411] In ADCC, cytotoxicity can be mediated by natural killer (NK) cells; however, macrophages, neutrophils and eosinophils can also mediate the cytotoxicity. In embodiments of the present invention, ADCC can include ADCC of the patient's CD16+ immune cells. In embodiments of the present invention, ADCC can include cells selected from ... ADCC of macrophages, neutrophils and eosinophils.

[0412] 5. The ligand according to any one of the preceding aspects, wherein the cytotoxicity is antibody-dependent cell-mediated cytotoxicity (ADCC), wherein the antibody is the ligand.

[0413] 6. The ligand according to any of the preceding aspects, wherein the ligand comprises an anti-CD7 antibody, an antibody fragment or a trap.

[0414] In an example, the ligand comprises a paired VH / VL anti-CD7 binding site, wherein VH and VL are human antibody variable domains. Additionally or alternatively, the antibody or fragment comprises a human Fc.

[0415] In examples, the ligand is a human ligand, such as a human antibody or fragment.

[0416] In an example, the ligand is capable of being internalized by a CD7+ cell. In an example, the ligand is capable of being internalized by a cancer cell. In an example, the ligand is capable of being internalized by a CEM cell in vitro.

[0417] 7. The ligand according to any one of the preceding aspects, wherein the patient has previously received a cancer chemotherapeutic agent.

[0418] In an example, the patient has previously received an immune checkpoint inhibitor, such as an antibody against an immune checkpoint inhibitor. In an example, the inhibitor is ipilimumab, nivolumab, pembrolizumab, or tremelimumab.

[0419] In examples, the patient has previously received anti-cancer radiation therapy.

[0420] 8. The ligand according to any of the preceding aspects, wherein the patient has previously received GCSF.

[0421] Chemotherapy can cause bone marrow suppression and low levels of white blood cells (neutropenia), making patients susceptible to infection and sepsis. GCSF stimulates the production of granulocytes (a type of white blood cell). In oncology and hematology, recombinant forms of GCSF are used in certain cancer patients to accelerate recovery from neutropenia after chemotherapy, allowing for more intensive treatment regimens. It can be administered to tumor patients by subcutaneous or intravenous routes. In the context of the present invention, administering GCSF simultaneously or sequentially (e.g., before) administering an anti-CD7 ligand to a patient may be beneficial in upregulating CD7 involved in CDC, ADCC, and ADCP-mediated CD7 signaling pathways of the present invention. + Cell type of cell killing.

[0422] 9. The ligand according to any one of the preceding aspects, wherein the patient has previously undergone chemotherapy and wherein immediately prior to administration of the ligand to the patient, the patient has received treatment for enhancing complement (eg, C1q) activity.

[0423] 10. The ligand according to any of the preceding aspects, wherein the patient has received administration of one or more complement components (eg, a composition comprising C1q).

[0424] For example, the components are included in blood or plasma that is administered to a patient.

[0425] 11. Comprising determining the level or activity of complement (eg, C1q) in the patient prior to administering said ligand.

[0426] For example, the C1q level is a patient's serum concentration in the range of 70 to 160 μg / ml, for example, as determined by a quantitative ELISA, such as a sandwich ELISA. Examples of suitable techniques for determination are listed in: Biotechnol J. 2009 Aug;4(8):1210-4. doi:10.1002 / biot.200800273, "Systemic lupus erythematosus and C1q: A quantitative ELISA for determining C1q levels in serum", Dillon SP et al. In an embodiment, the range is 100 to 160 μg / ml.

[0427] Normal complement levels in healthy people range from:

[0428] 1. Total complement level: 41-90 hemolytic units

[0429] 2. C1 level: 16-33 mg / dL

[0430] 3. C3 level: 88-252 mg / dL for men; 88-206 mg / dL for women

[0431] 4. C4 level: 12-72 mg / dL for men; 13-75 mg / dL for women

[0432] In an example, the invention comprises administering an anti-CD7 ligand with anti-CD46, anti-CD55 or anti-CD59 therapy to neutralize complement regulatory protein (CRP) function in a patient.

[0433] 12. comprising administering the ligand to the patient, thereby killing the CD7 + cancer cells, and the patient is then given chemotherapy.

[0434] 13. The ligand according to any one of the preceding aspects, wherein the cancer cell is an immune cell of the patient.

[0435] 14. The ligand according to any one of the preceding aspects, wherein the cancer cell is a T cell, a NK cell, a thymocyte or a bone marrow CD34 + CD38 - cell.

[0436] The cancer cell is, for example, CD7 + CD34 + CD2 T cells. The cancer cells are, for example, CD34 + CD38 - Immune cells (e.g., T cells). The cancer cells are, for example, CD34 + CD38 + Immune cells (eg, T cells).

[0437] 15. The ligand according to any one of the preceding aspects, wherein the T cell is an immature T cell.

[0438] Optionally, the immature T cells include one, two or more of the following types: DN1, DN2, DN3, DN4 and DP. DN1 cells are positive for the following markers: CD34, CD44, CD7, TdT, HLA-DR. DN2 cells are positive for the following markers: CD2, CD5, CD7, CD25, CD38, CD44, CD117, CD127, TdT, HLA-DR. DN3 cells are positive for the following markers: CD2, CD5, CD7, CD25, CD38, CD44, CD71, CD117, TdT. DN4 cells are positive for the following markers: CD1, CD2, CD5, CD7, CD38, TdT. DP cells are positive for the following markers: CD2, CD3, CD4 or CD8, CD7.

[0439] In an embodiment, the cancer cells include early thymic precursor (ETP) cells. The presence of such cells is associated with a high risk of leukemia and a low or no response to nelarabine. Thus, in an embodiment, the patient is refractory to nelarabine, for example, wherein the cancer cells include ETP cells.

[0440] In an example, the cancer cells are CD52+.

[0441] Optionally, the immature T cells are CD2 + 、CD5 + 、CD7 + .

[0442] 16. The ligand according to any one of the preceding aspects, wherein the T cell is a CD7 + CD34 + CD38 - T cells.

[0443] Optionally, the cell is, for example, a CD7 + CD34 + CD38 - Lin - T cells, such as wherein the cancer is AML.

[0444] Optionally, the T cells are CD8+ T cells. Wherein the T cells are CD4+ T cells.

[0445] Optionally, the cell comprises a plurality of cells, each cell in the plurality of cells comprising at least 100, 500 or 1000 copies of cell surface CD7 (see e.g., Figure 1 a, Aandahl, EM et al. J Immunol. 2003. 170: 2349-2355 for guidance on such determinations).

[0446] 17. The ligand according to any one of the preceding aspects, wherein the cell is a leukemia initiating cell (LIC).

[0447] 18. The ligand according to any one of the preceding aspects, wherein the cell is a leukemia cell, such as an AML or T-ALL cell.

[0448] 19. The ligand according to any one of the preceding aspects, wherein the cancer cells are T cells and NK cells are protected from killing in the patient.

[0449] 20. The ligand of any of the preceding aspects, wherein the ligand kills no more than 70% (e.g., no more than 80, 90, or 95%) of NK cells in a standard in vitro cell killing assay.

[0450] 21. The ligand according to any of the preceding aspects, wherein the Fc region is human Fc.

[0451] 22. The ligand according to any of the preceding aspects, wherein the Fc region is a wild-type Fc.

[0452] 23. The ligand according to any one of the preceding aspects, wherein the Fc region is a human IGHG1*01 or IGHG1*02 nucleotide sequence.

[0453] 24. The ligand of any one of the preceding aspects, wherein the cancer is acute myeloid leukemia (AML), T-ALL (e.g., patient-derived T-ALL), peripheral T-cell lymphoma (PTCL), or T-cell prolymphocytic leukemia (TPLL).

[0454] Optionally, AML is M1 / M2 AML. Optionally, the cancer is mixed lineage leukemia (MLL)-rearranged human acute lymphoblastic leukemia.

[0455] Optionally, the cancer is caused by CD7 + Immune cell (e.g., T cell) mediated cancers.

[0456] Optionally, the cancer is lymphocytic leukemia (LL) (eg, ALL or acute lymphoblastic leukemia), cutaneous T-cell lymphoma (CTCL), or melanoma. Optionally, the cancer is relapsed T-ALL or AML.

[0457] Optionally, the cancer is liver cancer. Optionally, the cancer is selected from melanoma, Merkel cell carcinoma, non-small cell lung cancer (squamous and non-squamous), renal cell carcinoma, bladder cancer, head and neck squamous cell carcinoma, mesothelioma, virus-induced cancers (such as cervical cancer and nasopharyngeal carcinoma), soft tissue sarcoma, hematological malignancies, such as Hodgkin's disease and non-Hodgkin's disease, and diffuse large B-cell lymphoma.

[0458] 25. The ligand according to any one of the preceding aspects, wherein the AML is CCAAT / enhancer binding protein alpha (CEBPA) mutated AML.

[0459] Optionally, the patient is homozygous for a CEBPA mutation.

[0460] 26. The ligand according to any one of the preceding aspects, wherein the patient has HLA-DR + CD7 + CD13 + CD14 - CD15 + CD33 + CD34 + The immunophenotype of, e.g., wherein the cancer is AML, e.g., CEBPAAML.

[0461] 27. The ligand according to any one of the preceding aspects, wherein the human is an adult.

[0462] For example, the adult is at least 18, 20, 30, 40, 50, 60, 70, 80, or 90 years old.

[0463] 28. The ligand according to any of the preceding aspects, wherein the human is an infant.

[0464] For example, the human is a child, such as a human under 18 years old, such as less than 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 year old.

[0465] 29. The ligand according to any of the preceding aspects, wherein the patient suffers a 1st or 2nd recurrence of the cancer, eg after previous treatment with a chemotherapeutic agent.

[0466] In examples, the agent includes an immune checkpoint inhibitor, such as those described herein.

[0467] 30. The ligand according to any one of the preceding aspects, wherein the patient has previously received nelarabine.

[0468] 31. The ligand according to any one of the preceding aspects, wherein the ligand and nelarabine are administered to the patient simultaneously or sequentially.

[0469] For example, the present invention may enable administration of lower doses than the standard of care of nelarabine.

[0470] 32. The ligand according to any one of the preceding aspects, wherein the ligand is administered to the patient simultaneously or sequentially with hematopoietic stem cell transplantation.

[0471] In examples, the transplant is an allogeneic transplant.

[0472] In an example, the ligand is administered no more frequently than once every 2 or 4 weeks. In an example, the ligand is administered every two weeks, monthly, or weekly.

[0473] 33. The ligand of any of the preceding aspects, wherein the treatment reduces cancer progression in the patient.

[0474] 34. The ligand of any of the preceding aspects, wherein the treatment increases cancer survival in a human patient.

[0475] 35. The ligand according to any one of the preceding aspects, wherein the treatment results in remission of cancer in the patient.

[0476] 36. The ligand according to any one of the preceding aspects, wherein the ligand mediates ADCP in a standard ADCP assay.

[0477] 37. The ligand of any preceding aspect, wherein the ligand mediates CDC killing of CEM cells in an in vitro CEM cell killing assay with an EC50 range of 10 to 500 pM (eg, 10 to 500 or 100 pM).

[0478] CEM cells are a well-known human T-ALL cell line. In an example, the CEM cells in the assay are in the presence of human complement (e.g., CEM cells are mixed with human serum including complement proteins). In an example, when evaluated against a control using non-cancerous human cells ("normal cells") instead of CEM cells, the ligand preferentially mediates killing of the cancer cells over normal cells. The normal cells may be cancer patient cells.

[0479] Optionally, in the assay, the ligand mediates the EC 50 of CEM cells and 90-100% killing of CEM cells.

[0480] 38. The ligand according to any one of the preceding aspects, wherein the ligand mediates ADCP killing of CEM cells in an in vitro CEM cell killing assay, wherein EC 50 The range is 10 to 500 pM (eg, 10 to 500 or 100 pM).

[0481] CEM cells are a well-known human T-ALL cell line. In an example, the CEM cells in the assay are in the presence of human complement (e.g., CEM cells are mixed with human serum including complement proteins). In an example, when evaluated against a control using non-cancerous human cells ("normal cells") instead of CEM cells, the ligand preferentially mediates killing of the cancer cells over normal cells. The normal cells may be cancer patient cells.

[0482] Optionally, in the assay, the ligand mediates the EC 50 of CEM cells and 90-100% killing of CEM cells.

[0483] In embodiments, the ligand mediates ADCC killing of CEM cells in an in vitro CEM cell killing assay, wherein EC 50 The range is 10 to 500 pM (eg, 10 to 500 or 100 pM).

[0484] CEM cells are a well-known human T-ALL cell line. In an example, the CEM cells in the assay are in the presence of human complement (e.g., CEM cells are mixed with human serum including complement proteins). In an example, when evaluated against a control using non-cancerous human cells ("normal cells") instead of CEM cells, the ligand preferentially mediates killing of the cancer cells over normal cells. The normal cells may be cancer patient cells.

[0485] Optionally, in the assay, the ligand mediates the EC 50 CEM cell killing and 90--100% CEM cell killing.

[0486] In an embodiment, the CEM ligand mediates cytotoxic killing of CEM cells in an in vitro CEM cell killing assay, wherein the EC 50 The range is 10 to 500 pM (eg, 10 to 500 or 100 pM).

[0487] CEM cells are a well-known human T-ALL cell line. In an example, the CEM cells in the assay are in the presence of human complement (e.g., CEM cells are mixed with human serum including complement proteins). In an example, when evaluated against a control using non-cancerous human cells ("normal cells") instead of CEM cells, the ligand preferentially mediates killing of the cancer cells over normal cells. The normal cells may be cancer patient cells.

[0488] Optionally, in the assay, the ligand mediates the EC 50 of CEM cells and 90-100% killing of CEM cells.

[0489] 39. The ligand of any one of the preceding aspects, wherein the ligand specifically binds to human CD7 and cynomolgus monkey CD7.

[0490] 40. The ligand according to any one of the preceding aspects, wherein the treatment does not produce cytokine storm syndrome in the patient.

[0491] 41. The ligand according to any one of the preceding aspects, wherein the cancer cell is a CD7 高 cell.

[0492] 42. A regimen for treating or preventing cancer in a human patient, wherein the regimen comprises performing the treatment described in any of the preceding aspects, wherein the ligand is an antibody and a first, second and third dose of the antibody is administered to the patient, wherein the doses are administered between 1 and 7 days apart, and wherein the sum of the doses is 0.1 to 100 mg / Kg of the antibody.

[0493] The present invention also provides a method of treating or preventing cancer in a human patient, the method comprising administering to the patient a ligand according to any one of the aspects.

[0494] The present invention also provides a method for detecting CD7 in a cell sample (such as a blood or serum sample). + A method for treating a cell, wherein the method comprises mixing the sample with a ligand of the present invention, whereby the ligand binds to CD7 + The ligand binds to the cells and the cells to which the ligand binds are detected or quantified.

[0495] Instead of or as an example of a "standard test," the test may be the method used in the examples herein.

[0496] Examples

[0497] To identify antibodies that usefully target human CD7 and can be used to treat cancers such as T-ALL.

[0498] A particularly desirable antibody, G09, when formatted as an IgG1 including the E430G mutation (also referred to as "G09 E430G"), exhibits human / cynomolgus CD7 cross-reactivity and provides efficient complement-dependent cytotoxicity (CDC)-dependent killing and potent macrophage-dependent phagocytic activity in vitro, as well as robust tumor cell depletion in a whole blood assay. This data set demonstrates antibodies including the G09 variable domain:

[0499] Shows potent CDC activity against most non-relapsed or relapsed T-ALL cells tested in vitro (EC50 = 50-500 pM; approximately 100% killing)

[0500] Demonstrated potent ADCP activity against relapsed T-ALL cells in vitro

[0501] Kills T-ALL cells in human whole blood assay (approximately 100%)

[0502] Kills tumors in the most severely immunocompromised mice (NSG)

[0503] Reduced potency in killing human peripheral T cells (50-75%) and NK cells (70-90%) in ex vivo assays.

[0504] Example 1

[0505] CD7 is expressed throughout the development of the T cell lineage, and is therefore expected to be expressed on all T-ALL primitive cells. It is recognized that based on CDC, antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP), the combination of immunoreactive CD7-IgG1 mAb is expected to lead to the depletion of these cells. Considering the importance of killing primitive cells, it is hoped to have a powerful depletion effect. It is recognized that the complex factor is that CD7 is expressed not only on T cells, but also on NK cells, so it is possible to deplete the effector cells of ADCC response. In addition, T-ALL malignant tumor patients receiving intensive chemotherapy may cause the effector cell level of mediated cytotoxicity to decrease. Therefore, it is recognized that effective depletion of T-ALL cells cannot be achieved only by ADCC during anti-CD7 treatment. Although the reduction of complement activity after chemotherapy is temporary in nature, complement activity seems to be effective for ALL patients during certain periods during therapy (15)( Fig.16 ).

[0506] We hypothesized that the efficacy of CD7-targeted therapy in patients with T-ALL malignancies could be enhanced by enhancing the potency of CD7-targeted cytotoxicity using strategies that increase CDC activity.

[0507] The classical pathway of CDC relies on the binding of C1q to cell surface antigen-bound antibodies. The ability of human antibodies to induce CDC is isotype-dependent, with the order of potency being IgM>>IgG3≥IgG1>>IgG2=IgG4. IgM and IgG3 are difficult to manufacture, making IgG1 the preferred Fc isotype for therapeutic antibody-mediated effects via CDC. Recently, several point mutations in the IgG1 CH3 domain, E345R, E430G, and other variants have been identified that, when bound to antigen, cause the immunoglobulin to form hexameric structures (16). These hexameric structures can enhance CDC activity by 2 to 3 orders of magnitude, inducing CDC at levels comparable to native IgM.

[0508] To evaluate these IgG1 variants, mutations were introduced into the CD7-specific benchmark antibody RFT2 (10, 17) and cultured in CCRF-CEM cells (referred to herein as CEM cells, available from ATCC as CCL-119 TM The mutations were evaluated on a CD7-expressing T-ALL cell line obtained using human serum as a complement source. The wild-type IgG1 version of the RFT2 antibody was found to have very limited or no killing activity on CEM cells, whereas the IgG1 variants of the three tested RFT2 antibodies mediated effective killing of CEM cells ( Figure 1 Among them, the killing activity of RFT2 E345R antibody was the most effective, with the maximum killing rate being almost 100%, and the EC 50 Less than 1nM.

[0509] The classical pathway of CDC is expected to be the primary MOA for the lead antibody. In addition, ADCP mediated by macrophages and phagocytosis mediated by neutrophils will also be evaluated. It was determined that the anti-CD7 antibody should bind to CD7 to initiate cell depletion, but the antibody should not activate the cells. Cytokine release using human whole blood was also evaluated for the lead antibody.

[0510] Methods and Materials

[0511] a) CDC assay

[0512] CCRF-CEM cells (referred to as CEM cells, CCL-119) or other T-ALL cells were 8.75×10 4Cells / ml were suspended in assay medium (RPMI1640, 10% hiFBS). Cell suspension was plated at 20 μl / well according to the plate map. Serially diluted antibodies were added at 10 μl / well according to the plate map. Human complement serum (Sigma, S1764) was reconstituted in 1 ml of ice-cold water. The reconstituted serum was diluted 1 to 3 in culture medium, and 10 ul of the diluted serum was added to each well of the plate. The plate was incubated at 37°C and 5% CO2 for 2 hours, equilibrated to room temperature for 15 minutes, and then 40 ul of the reconstituted serum was added to each well. (Promega). The plate was covered with an optical plate seal, agitated on a plate shaker at 300 rpm for 2 minutes to ensure all cells were lysed, and then used. The 384 protocol was read on an Envision plate reader. The luminescent signal generated by the mixture is proportional to the amount of ATP present. The maximum percent killing was calculated from triplicate and quadruplicate samples using the following equation:

[0513]

[0514] b) ADCP assay

[0515] Primary human macrophages were cultured in RPMI + 10% FBS supplemented with 100 ng / mL M-CSF (Peprotech) for 7-10 days. 5 Monocytes seeded at 10 cells / well in 12-well plates were differentiated to generate monocyte-derived macrophages (MDM). Macrophages were treated with 2 μM CellTrace TM CEM cell lines were maintained in RPMI + 10% FBS. For experiments using patient-derived T-ALL xenograft cells (PDTALL-39, -46, -47, and -Ad2R), stock solutions were recovered from frozen on the day of the assay. Normal primary T cells were freshly isolated from macrophage donor-matched peripheral blood mononuclear cells (PBMCs) by negative selection (using the human T cell isolation kit from Stemcell Technologies) immediately prior to the assay. Target cells (CEM, T-ALL, or normal T cells) were stained with 2 μM CellTrace TM CFSE (Thermo Fisher Scientific) labeled and resuspended in PBS.

[0516] Serial dilutions of anti-CD7 or control antibodies were prepared in 25 μL PBS at 2x final concentration. 25 μL of CFSE-labeled target cells were plated at 4 × 10 6 cells / mL (i.e. 1×10 5 Cells were pre-opsonized with different dilutions of anti-CD7 antibody in PBS. As controls, cells were mock opsonized (no antibody) or opsonized with appropriate dilutions of human IgG1 isotype control antibody. Cells were then washed once with excess assay medium including RPMI + 10% ultra-low IgG FBS (to eliminate unbound antibody) and resuspended to 5 × 10 cells in assay medium. 4 Aspirate the medium from the 12-well plate of CTV-labeled macrophages, and add 800 μL of the target cell suspension to duplicate wells (to give 4×10 4 Target cells / well and effectors: target ratio of 5:1) and incubated for 2.5 hours at 37°C, 5% CO2 to allow target cell phagocytosis.

[0517] Non-adherent cells were collected and combined with adherent macrophages and detached using cell digestion buffer (Life Technologies) and gentle cell scraping. Cells were then washed with PBS and stained with LIVE / DEAD fixable near-infrared (IR) dead cell dye (30 minutes at 4°C; Thermo Fisher Scientific) before being washed again with PBS and fixed in 4% paraformaldehyde (PFA; Affymetrix, USA) for 20 minutes at room temperature. Cells were resuspended in PBS containing 2mM EDTA for analysis. Single labeled cells and live / dead near-IR labeled ArC TM Amine reactive beads (Molecular Probes) were used for compensation. Flow cytometry acquisition was performed on an Attune NXT flow cytometer using 405, 488, and 637 lasers (Thermo Fisher Scientific), and data were analyzed using FlowJo v10.0.8rl (FlowJo LLC). Percent phagocytosis was calculated from duplicate samples using the following equation:

[0518]

[0519] c) Human whole blood assay

[0520] Regarding the use of anticoagulants, it was decided not to use standard heparin but to use low concentrations of hirudin, which had produced successful results in cynomolgus monkey studies. Plasma extracted from hirudin-treated blood mediated CDC of CEM cells in vitro, whereas plasma from heparinized blood did not (data not shown). Final concentrations of 1741G09 E430G, isotype control, rituximab, and ofatumumab were used in the study.

[0521] exist Whole blood cultures from three blood donors were set up and performed in tubes (Myriad RBM, USA). (TC) tubes were filled with freshly produced medium ± antibody / control. Tubes were stored at -20°C (<7 days) and used after thawing and thorough mixing (adjustment to room temperature). Within 60 minutes of blood collection, freshly drawn blood containing hirudin as an anticoagulant was transferred to tubes and incubate at 37 °C in a block thermostat for 20 h.

[0522] At the end of the culture period, immune cells were analyzed by flow cytometry. To accurately calculate cell counts, a defined volume of cultured whole blood was added to BD Tubes (CE, IDV). To determine immune status, a 7-color immunophenotyping panel (T cell CD45 + CD3 + NK cell CD45 + CD3 - CD16 + CD56 + ; B cell CD45 + CD3 - CD19 + ; Monocytes (CD14 + ). Cells were stained using a no-wash protocol from the antibody manufacturer (Miltenyi Biotec; 7-color immunophenotyping kit; #130-098-456). Briefly, at the end of the incubation period, The tube was centrifuged and 2 ml of The supernatant consisting of culture medium and plasma was used to thoroughly resuspend the blood cells. 50 μl of the resuspended immune cells were transferred to In a tube (containing a defined number of beads), add the detection antibody and incubate at 2-8°C for 10 minutes. Add hemolysis solution to eliminate erythrocytes (20 minutes at RT). Store the cells at 2-8°C before analysis by flow cytometry. TM Samples were acquired on a flow cytometer (BD Bioscience).

[0523] The data were analyzed by FlowJo (version 10.4.1; FlowJo LLC). The cells per volume were calculated according to the following equation:

[0524]

[0525] d) Antigen capture ELISA for quantification of serum 1741G09

[0526] 96-well high binding plates were coated overnight at 4°C with 50 μl of recombinant soluble human CD7 protein (Sino Biologicals, 11028-H08H) diluted in PBS at 2 μg / ml. The plates were washed with PBS+0.1% Tween at 3×300 μl / well using a plate (wash buffer) washer, each well was blocked with 200 μl PBS+1% BSA for at least 1 hour at room temperature, and then washed again. Serially diluted standards, samples, and controls were added to the plate at 50 μl / well. After incubation at room temperature with shaking at 300 rpm for 1 hour, the plate was washed with washing buffer at 5×300 μl / well. HRP-conjugated anti-human IgG diluted in PBS+1% BSA at 50 μl / well was added. After incubation at room temperature with shaking at 300 rpm for 1 hour, the plate was washed with washing buffer at 5×300 μl / well. Plates were added with 50 μl / well of TMB substrate, incubated for 30 minutes in the dark at room temperature, and then 50 μl / well stop solution was added. Optical density was determined within 5 minutes using a microplate reader set to 450 nm and calibrated at 640 nm. Data were imported into Softmax Pro and regressed using 4PL curve fitting with a weighting factor of 1 / y.

[0527] e) Cytokine release assay

[0528] High binding plates were coated with 1741G09 E430G and other control antibodies at a concentration series and incubated overnight at room temperature with the lid open to air dry. Plates were washed with PBS, blocked with cell culture medium by standing for 30 minutes, and then aspirated just before adding cells.

[0529] Warm culture medium was added dropwise to the vial with cryopreserved human PBMCs, and the cells were then cultured at high density (0.5-1×10 7 After pre-incubation, PBMCs were seeded into 96-well polypropylene plates with 240 μl / well of culture medium (RPMI 1640, 10% FBS, 1% penicillin / streptomycin, 2 mM L-glutamine) and then incubated for 1 hour at 37°C 5% CO2. PBMC cell cultures (200 μl / well, 2×105 Cells / well) were transferred to the fixed test reagent in the prepared plate and cultured for 48 hours at 37°C, 5% CO2. After the cell culture supernatant was collected and stored at -80°C until required for analysis, the plate was centrifuged at 200g for 10 minutes.

[0530] Luminex assessment of cytokine levels in cell culture supernatants was performed following the manufacturer's protocol. The induced levels of each cytokine were interpolated from the standard curve using a 5-point nonlinear regression analysis. The interpolated data were then normalized to the unstimulated control.

[0531] f) Transient protein expression

[0532] Transient protein expression was performed. Cultures were harvested on day 12 post-transfection and the clarified harvest was transferred to the purification team.

[0533] g) Stable pool protein expression

[0534] Stable pool protein expression was performed.

[0535] Discover

[0536] a) Leader generation

[0537] The discovery work of the anti-CD7 program consists of immunization, hybridoma generation, antibody screening, biological evaluation of antibody potency and biophysical characterization, e.g. Figure 2 shown.

[0538] Evaluation of drug toxicology requires antibodies that are cross-reactive to human and cynomolgus monkey CD7 antigens. Human and cynomolgus monkey CD7 proteins share only 86% identity. To ensure this cross-reactivity, Kymice (26) was co-immunized with human and cynomolgus monkey antigens. Titers were examined by flow cytometric analysis, in which the degree of binding of polyclonal sera to human and cynomolgus monkey CD7-expressing CHO cells was quantified. When sera were diluted more than 10 4 Mice with detectable binding titers at the dilution were used for hybridoma generation.

[0539] There were four selection criteria for the hits in the primary, secondary and tertiary in vitro screens: (i) high-level binding to human CD7, (ii) specificity for both human and cynomolgus CD7, (iii) efficient T-ALL cell depletion and (iv) inability to induce cytokine release from T cells.

[0540] Hybridoma supernatants were used for primary and secondary screening to identify binders. The primary screen consisted of a high-throughput LiCOR-based cell binding assay to include all potential cross-reactive binders. A secondary screen using flow cytometry was used to confirm cross-reactivity. Results were presented in Figure 3 The binding of human and cynomolgus CD7 to CEM cells and recombinant cynomolgus CD7 CHO cells was quantified by geometric mean fluorescence intensity (GEM) using flow cytometry. CEM cells are a human T-ALL cell line expressing endogenous CD7.

[0541] Seven hits that showed strong and cross-reactive binding to human and cynomolgus CD7 were further examined by measuring SPR to understand their relative binding to the two antigens. Five leads (1730C2, 1734F05, 1738B07, 1741G09, 1896A03) were confirmed to show similar binding to CD7 from both species, as shown in Table 1. Figure 4 and as shown in Table 1. The data were compared with the benchmark RFT2 IgG1 (a chimeric antibody having mouse variable regions and human constant regions).

[0542] The hit DNA sequences were retrieved from the hybridoma clones and the constant region was reformatted into human IgG1 with the E345R variant. The E345R variant in the IgG1 CH3 region has been shown to enhance complement dependent cytotoxicity (CDC) activity. Figure 1 As shown, RFT2 IgG1 E345R but not RFT2 IgG1 can effectively kill CEM cells in the CDC assay ( Figure 1 ). The IgG1 E345R reformatted antibody was expressed and tested in a CDC assay using human serum as complement source and CEM cells as target cells. Figure 5 In Table 2, two leads 1741E04 E345R and 1741G09 E345R were shown to have 100% maximal killing and potency below 200 pM. The former only binds to human antigens, while the latter binds to both human and cynomolgus monkey antigens. These leads were also generated with wild-type IgG1 and tested in the CDC assay. No significant killing was observed even at concentrations above 10 nM (data not shown).

[0543] To evaluate whether the three leads can mediate phagocytosis by macrophages, they were tested in an ADCP assay using macrophages derived from peripheral blood monocytes as the source of phagocytic cells and CEM cells as target cells. The lead 1741G09 E345R, which showed the best activity in the CDC assay, also showed the best activity among the three leads in this assay, as shown in Table 1. Figure 6 shown.

[0544] It is generally believed that dual staining correlates with internalization of the target cell into the effector cell (or at least localization within the phagocytic cup). In the experiments, images of the cells were captured at the endpoint of the phagocytosis assay using Amnis Imagestream (Merck Millipore) and analyzed using software tools that allow differentiation between internalized and extracellularly bound particles to demonstrate phagocytosis of the target cells. Similar techniques have been described elsewhere for this purpose (27). In addition, cell harvesting by enzymatic separation using cell digestion fluid at the assay endpoint may disrupt cell-cell contacts, which could be misinterpreted as a phagocytosis (internalization) event.

[0545] During the screening process, there was another variant IgG1 E430G that was also reported to enhance CDC activity (28). In the test, the 1741G09 E430G variant had similar killing efficacy to the 1741G09 E345R variant (Figure 7.A.). The half-life of 1741G09E430 in NSG mice was 136.7 hours (Figure 7.B.), which is within the normal range for human IgG1 in such mice. However, the half-life of 1741G09 E345R was much shorter, only 20.9 hours, which may be due to the higher aggregation tendency of this variant. Based on this observation, 1741G09 E430G was used instead of 1741G09 E345R as the lead molecule, and the CDC and ADCP activities of this molecule were further evaluated.

[0546] b) In vitro precursor distribution

[0547] Antibody discovery activity is based on the half-life of combination, cell depletion assay and molecule, and lead antibody 1741G09E430G is identified.In order to assess its killing activity to different T-ALL cells, 14 kinds of different T-ALL cell lines of leading are tested in CDC assay, and described cell line comprises two kinds of commercial in vitro cell lines (CEM and HSB2), six in vivo passage non-relapse cell lines (PDTALL8, PDTALL11, PDTALL12, PDTALL13, PDTALL16 and PDTALL18) and six in vivo passage relapse cell lines (PDTALL39, PDTALL46, PDTALL47, PDTALL51R, PDTALLAd2R and PDTALLAd4) (29).The clinical and gene spectrum of these cell lines are shown in Table 7. 1741G09 E430G was shown to effectively kill the majority of cell lines (11 of 14) (Figure 8), with potencies ranging from approximately 50-500 pM (Table 3).

[0548] In the assays performed, potency and maximum killing correlated with cell surface CD7 expression (Tables 8 and 9). Cell lines can be ranked based on the level of CD7 expression on the cell surface, as shown in Table 3. Reflecting the expression level, PDTALL47, HSB, PDTALLAd4, PDTALL51R, PDTALL39, PDTALL8, CEM, and PDTALL16, which have high or intermediate CD7 expression levels (relative to CEM cells), have the highest maximum killing, approaching 100%, and EC 50 The values ​​were 50 pM to 200 pM; PDATLLAd2R, PDATALL12, and PDTALL11 with moderate to low expression levels had a maximum killing of nearly 90%, and the EC 50 The maximum killing of PDTALL13 and PDTALL18, which were expressed at much lower levels, was less than 70%, and the EC 50 The levels of PDTALL46 and PDTALL46 were higher than those of PDTALL46 and PDTALL2R, respectively. Both cell lines had similar EC values ​​close to 350 pM. 50 , but the maximum killing of PDTALL46 was lower than that of PDTALLAd2R (78% vs. 93%), probably because the expression level of complement regulatory proteins (CRP: CD46, CD52 and CD59) of the former was higher than that of the latter (Table 9). It is reported that CRP is used as an antagonist against complement activity on the cell surface. In summary, these results indicate that the antibody efficacy of CDC activity depends mainly on the expression of target antigens on the cell surface and can be regulated by CRP.

[0549] like Fig. 9 As shown, the lead was also tested in an ADCP assay using different T-ALL cells. Macrophages derived from human peripheral monocytes were differentiated and used to evaluate the ADCP activity of the 1741G09 E430G molecule on T-ALL cell lines. Of the five cell lines tested, four showed close to 100% macrophage phagocytosis. In the CDC assay, 1741G09E430G only depleted 80% of the relapse cell line PDTALL46 cells. However, it mediated almost 100% of the phagocytosis of the same cells in the ADCP assay. On the other hand, in the ADCP assay, 1741G09 E430G only mediated 75% of the other relapse cell line PDTALLad2R cells. However, in the CDC assay, 1741G09 E430G depleted nearly 95% of the same cells. These data support the theory that multiple MOAs can complement each other and mediate the effectiveness of T-ALL cells.

[0550] c) Cytokine release distribution

[0551] Since CD7 is expressed on peripheral T and NK cells, antibody ligation may lead to activation of these cells. It has been reported that anti-CD7 mAbs can have mitogenic effects, increase calcium flux and increase IL-2 production (30). However, in previous clinical trials of renal transplantation therapy, the anti-CD7 mAb, RFT-2, did not cause any significant concerns about cytokine storm (10). To evaluate the ability of the 1741G09 E430G antibody to stimulate human peripheral blood mononuclear cells (PBMCs), the effect of the molecule on cytokine release from PBMCs was tested after air-drying and fixation.

[0552] 1741G09 E430G was evaluated in PBMCs from five individual donors as measured by the release of specific cytokines and chemokines. Corresponding isotype controls were used to monitor nonspecific activation of PBMC cultures. Superagonist anti-CD28 and anti-CD3 (OKT3) antibodies were used as positive controls.

[0553] In the cytokine panel, six cytokines were slightly increased in samples treated with 1741G09E430G (IL8, 2.8-fold; MIP-1α, 2.9-fold; TNFα, 4.2-fold; IL1β, 4.6-fold; IL6, 1.7-fold) when compared to samples treated with the highest tested concentration (60 μg / ml) of IgG1. The IgG1 E430G isotype control also slightly increased the release of these cytokines, suggesting that the increase may not be CD7 specific. The positive control anti-CD3 (clone OKT3) and superagonist anti-CD28 induced a characteristic cytokine profile for all donors, confirming that the assay performed as expected, with a much higher general induction (1.0-fold) than any of the test articles or isotype controls. Fig.10 and Table 4).

[0554] Discovery Activity Summary

[0555] Seven leads were selected from primary and secondary cell binding screens. Five were confirmed to have cross-reactivity with human and cynomolgus CD7 proteins by SPR measurement of binding to soluble CD7. The five leads were reformatted into human IgG1 E345RFc and tested in CDC and ADCP assays in CEM cells. The 1741G09 E345R antibody was selected as the lead molecule because it had the most potent CDC and ADCP activity. Considering that this variant had a better half-life in vivo, the lead antibody was further reformatted into IgG1 E430G Fc. The potent CDC activity of the 1741G9 E430G antibody was also demonstrated in different T-ALL cell lines, with a maximum killing of 100% for most cell lines and strong potency (EC 50=50-500 pM). 1741G09 E430G mAb did not significantly increase cytokine release from PBMCs when compared to superagonists, anti-CD3 or anti-CD28.

[0556] c) Biological evaluation

[0557] In vitro CDC assay on PBMC T cells and NK cells

[0558] Peripheral T cells and NK cells are susceptible to leader molecule-mediated depletion because they express CD7. Peripheral T cells and NK cells were isolated from two different donors and used to evaluate the CDC activity of 1741G09 E430G. Although no significant T cell depletion was detected ( Fig.11 .A.), but with a maximum of 65% NK cell depletion, where EC 50 Close to 300pM( Fig.11 .B.) In EC 50 values, and T-ALL cell depletion was observed (Table 2). Notably, NK cells had higher CD7 expression and lower CRP expression compared with T cells ( Fig.17 ).

[0559] In vitro human whole blood assay

[0560] To assess whether 1741G09 could deplete normal peripheral T cells and NK cells in a more physiological context, we used a The effect of the antibody on human whole blood status was evaluated using an assay. The 1741G09 E430G antibody was used at concentrations ranging from 0.01 to 100 μg / ml and compared to an isotype control (100 μg / ml). Two positive controls, rituximab and ofatumumab (100 μg / ml), were also included. Whole blood and antibodies were incubated for 20 hours before evaluating cell depletion. Three human donors were used in the assay, referred to as A, B, and C. The number of cell subsets per μl of sample was determined by flow cytometry. The results are shown in Fig.12 The measurements were performed by Hotscreen.

[0561] In the four cell types measured (B, T, NK cells and monocytes), the cultures treated with isotype controls showed cell counts very similar to the negative control. Ofatumumab and rituximab are therapeutic antibodies targeting B cells, and include positive controls as specific cell depletion. In fact, B cells were almost completely eliminated throughout the culture process. The concentration-dependent depletion of T cells and NK cells by 1741G09 E430G was observed in all three donors. Compared with negative (no antibody) or isotype controls, the number of T cells and NK cells decreased. 1741G09 E430G antibodies depleted up to 90% of NK cells and up to 75% of T cells. Compared with T cell depletion, the antibody is more effective for NK depletion. Under the 1741G09 E430G concentration of 0.1 μg / ml (≈0.67nM), NK cells rather than T cells were significantly depleted.

[0562] The reduction of T cells seems to be related to CD4 + With CD8 + The ratio between T cells was not affected (data not shown). B cell and monocyte counts did not change significantly, indicating that 1741G09 E430G is specific for its cell depleting activity.

[0563] Up to 65% NK cell depletion was observed in CDC assays using human serum and isolated NK or T cells, but no significant T cell depletion ( Fig.11 ). Determination from human whole blood ( Fig.12 The higher maximal depletion rates obtained in the 247 cells (90% for NK cells and 75% for T cells) suggest the presence of additional effector cell components involved in cell depletion in this assay.

[0564] Ex vivo human whole blood assay spiked with T-ALL cells

[0565] Ideally, tumor cell depletion should be assessed in blood samples of T-ALL. However, since there are few patients and primary T-ALL cells are difficult to culture, it is difficult to obtain such samples. To circumvent this problem, the T-ALL cell line, CEM cells, were spiked into blood samples from healthy donors. The survival rate of CEM cells and the survival rate of NK cells, T cells and B cells from healthy donors were assessed in the presence of 10 μg / ml (≈67nM) of 1741G09 IgG1 E430G, 1741G09 IgG1 wild type, E430G IgG1 isotype control or ofatumumab. High concentrations of antibodies were used to ensure maximum killing. The results are shown in Fig.13 middle.

[0566] 1741G09 E430G significantly reduces CEM cell counts in spiked whole blood samples. 1741G09wt antibody was included as a comparison with the CDC enhanced version. Unlike 1741G09 E430G antibody, the wtFc version of 1741G09 has been shown to not significantly deplete CEM cells in CDC assays (data not shown). In whole blood assays, both wt and E430G versions of 1741G09 significantly reduced the number of CEM cells, but the efficacy of the E430G version was much greater (average depletion percentage 96.2% vs. 66.8%). The depletion of CEM cells by 1741G09 wt antibody indicates that other effector components other than CDC are also involved in the process. As expected, aufam significantly reduced B cell counts without affecting CEM, NK or T cell counts.

[0567] NK and T cell counts were also significantly reduced by 1741G09 E430G, but not 1741G09 wild-type. Depletion of NK cells (75%) and T cells (52%) was not as high as depletion of CEM cells (96%). This data confirms that the effect of the 1741G09 E430G antibody is related to the CD7 expression level of the target cells (CEM cells>NK cells>T cells, Figure 21), and that its effect on cell depletion is greater than that of the wild-type Fc version.

[0568] Effect of anti-C5a antibody on leader-induced cell depletion

[0569] To better understand the lead mode of action, the effects of anti-complement 5a (C5a) antibodies on 1741G09 E430G, 1741G09 wild-type, and ofatumumab-induced cell depletion were evaluated. The complement pathway can be divided into the activation pathway and the lytic pathway ( Fig.18 )(31). C5 can be cleaved into C5a (activation pathway) and C5b (cleavage pathway) by C3. C5a has chemotactic and anaphylactic properties and plays an important role in innate immune responses (smooth muscle contraction, vascular permeability, degranulation of mast cells and basophils, directional migration of neutrophils, eosinophils, basophils and monocytes). The antibodies used in the assay of the present invention are able to inhibit the binding of C5a to the C5a receptor without blocking the cleavage of C5 (32).

[0570] Both the wt and E430G versions of 1741G09 reduced CEM cell counts by 49% and 90%, respectively ( Fig.14). Addition of anti-C5a antibody did not affect CEM cell depletion induced by either version. Although anti-C5a did not block CEM cell depletion, the MOA behind the two versions of 1741G09 would be expected to be quite different. The lack of inhibition of CEM depletion by 1741G09 E430G supports the hypothesis that this antibody acts through a lytic pathway. E430G-driven depletion is unlikely to be attributable to NK cell-mediated ADCC, as NK cells are rapidly depleted through CDC once 1741G09 E430G is added. In contrast, the wt version did not deplete NK or CEM cells through CDC (data not shown). Therefore, depletion of CEM cells by the wt version could be through NK cell-mediated ADCC, macrophage-mediated phagocytosis, or neutrophil-mediated phagocytosis, none of which appear to be inhibited by anti-C5a antibodies in this experimental setting.

[0571] The 1741G09 E430G antibody reduced the counts of NK and T cells (70% and 49%, respectively). Addition of anti-C5a restored the 1741G09 E430G-induced T cell reduction to cell counts similar to the isotype control compared to CEM cells ( Fig.14 ), indicating that the depletion of T cells by 1741G09 E430G is not due to the classical or lytic pathway, but to the inflammatory pathway of complement depletion. This is consistent with the results of CDC assays in which 1741G09 E430G did not significantly deplete PBMC T cells. Addition of anti-C5a also partially restored the 1741G09 E430G-induced NK cell reduction, indicating that this molecule depletes NK cells through both the activation pathway and the lytic pathway. The effect of ofatumumab may be partially reversed by anti-C5a; however, it should be noted that this experiment was performed in only one donor. Incubation of whole blood with 1741G09 in the presence of a lytic pathway inhibitor (such as eculizumab) will confirm the requirement for this pathway.

[0572] In vivo xenograft studies

[0573] Anti-CD7 antibodies have previously been shown to be effective in xenograft models (33). To evaluate the efficacy of the 1741G09 E430G antibody, the antibody was tested in a pediatric relapsed PDX T-ALL xenograft model.

[0574] On day 0, 5 × 10 6After 100 PDTALL46 cells were added, NSG mice were dosed three times a week at 10 mg / kg from day 3 until the end of the study. Blood was drawn at serial time points to assess the expression level of human CD5 in the blood by flow cytometry, because no anti-CD7 antibodies that did not compete with 1741G09 were identified. The Kaplan-Meier plot demonstrated that the survival time of the group treated with 1741G09 E345R was significantly increased compared to the group with isotype control ( Fig.15 Since NSG mice are severely immunodeficient, lacking T cells, B cells, and NK cells, and also have defects in complement activity and macrophages, neutrophil-mediated phagocytosis (34) in the presence of 1741G09E430G may mediate the depletion observed in this mouse model.

[0575] Developability

[0576] As part of the developability assessment of candidate 1741G09 E430G, three pre-CMC studies have been conducted: early formulation screening, accelerated / real-time studies, and forced degradation studies.

[0577] In early formulation screening, candidates were dissolved in 12+ different platform formulation buffers and then evaluated for colloidal and conformational stability (T m and T agg Determined by intrinsic fluorescence and SLS, respectively). Two platform buffers demonstrated suitable stability and were shortlisted for accelerated and real-time studies: Candidates were prepared at 1 mg / ml in each of the two formulation buffers, or PBS as an additional control, and incubated for two weeks at 5°C, 25°C, and 37°C. Experiments were performed in triplicate for each condition. The following quality attributes were measured: aggregation by SEC-HPLC and DLS, fragmentation by SDS-PAGE, activity by CDC functional assay. After two weeks of incubation under any of the conditions tested, no significant changes in any quality attribute or activity were observed. Additionally, candidates were subjected to freeze / thaw stress and no changes in the same quality attributes were observed after 3 cycles of storage at -70°C for at least 18 hours followed by thawing for 3 hours at room temperature.

[0578] Finally, forced degradation studies were performed on the candidates at 1 mg / ml in PBS: forced deamidation (incubation in 1% ammonium bicarbonate at 37°C for 72 hours), forced oxidation (incubation in 0.03%, 0.003% and 0.0003% H2O2 at 25°C for 24 hours), and acidic hold (incubation at 25°C and pH 2.8 for 3 hours). The activity of the CDC functional assay was tested in duplicate before and after stress conditions. No significant changes were observed after forced deamidation, acid hold and forced oxidation at low and medium levels of H2O2, while oxidation at higher H2O2 concentrations showed an 87% decrease in activity and an EC 50 Increased 4 times.

[0579] Consistent with in silico predictions, experimental data suggest low risk from a developability perspective: candidates can be purified by a platform Protein A process with suitable product quality (less than 1% aggregates, nominal concentration of 10 mg / ml into PBS pH 7.4 as platform buffer). Early formulation screening demonstrated that the platform formulation buffer could further improve colloidal and conformational stability, while accelerated and real-time conditions after two weeks did not impact product quality or activity. Force degradation testing highlighted low risk overall, with no impact due to freeze / thaw, acidic hold, deamidation; oxidation risk was seen as low / moderate and could be managed by formulation with addition of sacrificial antioxidant excipients.

[0580] Overall, based on the computational and experimental datasets presented here, the candidates present a low risk from a pre-CMC perspective.

[0581] Lead molecule expression

[0582] The lead molecule in the CD7 project, 1741G09 HuIgG1 E430G, C-terminal lysine truncated, Phe variant of the light chain (1741G09 E430G), has been expressed in both transient and stable pool expression systems.

[0583] 1741G09 E430G was expressed in three different ratios in a transient system: 30 ml, 200 ml, and 2 L. There were no significant differences in cell growth or viability compared to cultures expressing a control antibody. On day 5 post-transfection, expression levels were the same in all ratios, while expression was different on day 12, with expression levels >600 mg / L in the 30 ml and 2 L ratios. Expression plateaued at the 200 ml ratio. This difference in expression yield at different ratios is not uncommon.

[0584] The expression yield of 1741G09 E430G at 30ml and 2L ratios was higher than the standard high expression control antibody expressed with all molecules ( Fig.18). This is a good initial indicator that 1741G09 E430G can be expressed at appropriate levels. However, it is important to note that although anecdotal evidence suggests that if a molecule is highly expressed in a transient system, then said molecule may produce a high expressing stable cell line, the predictability of stable results from transient systems has not been fully evaluated.

[0585] Three pools of cells stably expressing 1741G09 E430G have been generated. On day 13 of fed-batch overgrowth, the average expression of these pools was 385 mg / L. The 1741G09E430G stable pool expression was 43% of the expression observed in the high-expressing control antibody pool generated in parallel. The pool may contain a mixture of high- and low-expressing clones, and therefore selecting the appropriate clone may result in good yields. In fact, for unrelated molecules, mini-pools were generated from pools that expressed about 50% of the control and achieved >5-fold expression improvements.

[0586] References

[0587] 1. Ware RE, Haynes BF. T cell CD7 mRNA expression is regulated by both transcriptional and post-transcriptional mechanisms. Int Immunol. 1993 Feb;5(2):179-87.

[0588] 2. Barcena A, Muench MO, Galy AH, Cupp J, Roncarolo MG, Phillips JH, et al. Phenotypic and functional analysis of T-cell precursors in the human fetal liver and thymus: CD7 expression in the early stages of T-and myeloid-cell development. Blood. 1993 Dec 1;82(11):3401-14.

[0589] 3.Hao QL, George AA, Zhu J, Barsky L, Zielinska E, Wang X, et al. Human intrathymic lineage commitment is marked by differential CD7 expression: identification of CD7-lympho-myeloid thymic progenitors. Blood. 2008 Feb 1;111(3):1318-26.

[0590] 4. Reinhold U, Abken H. CD4+CD7- T cells: a separate subpopulation of memory T cells? J Clin Immunol. 1997 Jul;17(4):265-71.

[0591] 5. Ware RE, Hart MK, Haynes BF. Induction of T cell CD7 gene transcription by nonmitogenicionomycin-induced transmembrane calcium flux. J Immunol. Baltimore, MD 1950. 1991 Oct 15;147(8):2787-94.

[0592] 6. Rabinowich H, Lin WC, Herberman RB, Whiteside TL. Signaling via CD7 molecules on human NK cells. Induction of tyrosine phosphorylation and beta l integrin-mediated adhesion to fibronectin. J Immunol. Baltimore, MD 1950. 1994 Oct 15;153(8):3504-13.

[0593] 7. Lee DM, Staats HF, Sundy JS, Patel DD, Sempowski GD, Scearce RM, et al. Immunologic characterization of CD7-deficient mice. J Immunol. Baltimore, MD 1950. 1998 Jun 15;160(12):5749-56.

[0594] 8. Sempowski GD, Lee DM, Scearce RM, Patel DD, Haynes BF. Resistance of CD7-deficient mice to lipopolysaccharide-induced shock syndromes. J Exp Med. 1999 Mar 15;189(6):1011-6.

[0595] 9. Carriere D, Arcier JM, Derocq JM, Fontaine C, Richer G. Antigenic modulation induced by four monoclonal antibodies adsorbed on gold particles (specificity anti-CD4, anti-CD5, anti-CD7, and anti-150-kDa antigen): Relationship between modulation and cytotoxic activity of immunotoxins. Exp Cell Res. 1989 May;182(1):114-28.

[0596] 10. Sharma LC, Muirhead N, Lazarovits AI. Human mouse chimeric CD7 monoclonal antibody (SDZCHH380) for the prophylaxis of kidney transplant rejection: analysis beyond 4 years. Transplant Proc. 1997 Mar;29(1-2):323-4.

[0597] 11. Rappl G, Abken H, Muche JM, Sterry W, Tilgen W, Andre S, et al. CD4+CD7-leukemic T cells from patients with Sezary syndrome are protected from galectin-1-triggered T cell death. Leukemia. 2002 May;16(5):840-5.

[0598] 12. Png YT, Vinanica N, Kamiya T, Shimasaki N, Coustan-Smith E, Campana D. Blockade of CD7 expression in T cells for effective chimeric antigen receptor targeting of T-cell malignancies. Blood Adv. 2017 Nov 28;1(25):2348-60.

[0599] 13. Kita K, Miwa H, Nakase K, Kawakami K, Kobayashi T, Shirakawa S, et al. Clinical importance of CD7 expression in acute myelocytic leukemia. The Japan Cooperative Group of Leukemia / Lymphoma. Blood. 1993 May 1;81(9):2399-405.

[0600] 14. Rohrs S, Scherr M, Romani J, Zaborski M, Drexler HG, Quentmeier H. CD7 in acute myeloid leukemia: correlation with loss of wild-type CEBPA, consequences of epigenetic regulation. J Hematol Oncol. 2010 Apr 14;3:15.

[0601] 15 Keizer MP, Kamp AM, Aarts C, Geisler J, Caron HN, van de Wetering MD, et al. The High Prevalence of Functional Complement Defects Induced by Chemotherapy. Front Immunol. 2016;7:420.

[0602] 16. Diebolder CA, Beurskens FJ, de Jong RN, Koning RI, Strumane K, Lindorfer MA, et al. Complement is activated by IgG hexamers assembled at the cell surface. Science. 2014 Mar 14;343(6176):1260-3.

[0603] 17. Heinrich G, Gram H, Kocher HP, Schreier MH, Ryffel B, Akbar A, et al. Characterization of a human T cell-specific chimeric antibody (CD7) with human constant and mouse variable regions. J Immunol. Baltimore, MD 1950. 1989 Dec 1;143(11):3589–97.

[0604] 18 Ravandi F, Aribi A, O'Brien S, Faderl S, Jones D, Ferrajoli A, et al. Phase II study of alemtuzumab in combination with pentostatin in patients with. J Clin Oncol Off J Am Soc Clin Oncol. 2009 Nov 10;27(32):5425-30.

[0605] 19. Cooper ML, Choi J, Staser K, Ritchey JK, Devenport JM, Eckardt K, et al. An “off-the-shelf” fratricide-resistant CAR-T for the treatment of T cell hematologic malignancies. Leukemia, February 20, 2018;

[0606] 20. Haapaniemi E, Botla S, Persson J, Schmierer B, Taipale J. CRISPR-Cas9 genome editing induces a p53-mediated DNA damage response. Nat Med. 2018 Jul;24(7):927-30.

[0607] 21. Kosicki M, Tomberg K, Bradley A. Repair of double-strand breaks induced by CRISPR-Cas9 leads to large deletions and complex rearrangements. Nat Biotechnol. 2018 Sep;36(8):765-71.

[0608] 22. Van Vlierberghe P, Ferrando A. The molecular basis of T cell acute lymphoblastic leukemia. J Clin Invest. 2012 Oct;122(10):3398-406.

[0609] 23. Coustan-Smith E, Mullighan CG, Onciu M, Behm FG, Raimondi SC, Pei D, et al. Early T-cell precursor leukaemia: a subtype of very high-risk acute lymphoblastic leukaemia. Lancet Oncol. 2009 Feb;10(2):147–56.

[0610] 24.Marks DI, Paietta EM, Moorman AV, Richards SM, Buck G, DeWald G, et al. T-cell acute lymphoblastic leukemia in adults: clinical features, immunophenotype, cytogenetics, and outcome from the larger randomized prospective trial (UKALL XII / ECOG 2993). Blood. 2009 Dec 10;114(25):5136-45.

[0611] 25.Aldoss I, Bargou RC, Nagorsen D, Friberg GR, Baeuerle PA, Forman SJ. Redirecting T cells toeradicate B-cell acute lymphoblastic leukemia: bispecific. Leukemia. 2017 Apr;31(4):777-87.

[0612] 26. Lee EC, Liang Q, Ali H, Bayliss L, Beasley A, Bloomfield-Gerdes T, et al. Complete humanization of the mouse immunoglobulin loci enables efficient therapeutic antibody discovery. Nature Biotechnology. 2014 Apr;32(4):356-63.

[0613] 27. Phanse Y, Ramer-Tait AE, Friend SL, Carrillo-Conde B, Lueth P, Oster CJ, et al. Analyzing cellular internalization of nanoparticles and bacteria by multi-spectral imaging flow cytometry. USA; 2012.

[0614] 28. de Jong RN, Beurskens FJ, Verploegen S, Strumane K, van Kampen MD, Voorhorst M, et al. A Novel Platform for the Potentiation of Therapeutic Antibodies Based on Antigen-Dependent Formation of IgG Hexamers at the Cell Surface. PLoS Biol. 2016 Jan;14(1):e1002344.

[0615] 29. Agnusdei V, Minuzzo S, Frasson C, Grassi A, Axelrod F, Satyal S, et al. Therapeutic antibody targeting of Notchl in T-acute lymphoblastic leukemia xenografts. Leukemia. 2014 Feb;28(2):278-88.

[0616] 30. Jung LK, Roy AK, Chakkalath HR. CD7 augments T cell proliferation via the interleukin-2 autocrine pathway. Cell Immunol. 1992 Apr 15;141(1):189-99.

[0617] 31. Markiewski MM, Lambris JD. The role of complement in inflammatory diseases from behind the scenes into the spotlight. Am J Pathol. 2007 Sep;171(3):715-27.

[0618] 32. Kola A, Baensch M, Bautsch W, Hennecke M, Klos A, Casaretto M, et al. Epitope mapping of a C5a neutralizing mAb using a combined approach of phage display, synthetic peptides and site-directed mutagenesis. Immunotechnology Int J Immunol Eng. 1996 Jun;2(2):1115-26.

[0619] 33. Baum W, Steininger H, Bair HJ, Becker W, Hansen-Hagge TE, Kressel M, et al. Therapy with CD7 monoclonal antibody TH-69 is highly effective for xenografied human T-cell ALL. Br J Haematol. 1996 Nov;95(2):327-38.

[0620] 34. Matlung HL, Babes L, Zhao XW, van Houdt M, Treffers LW, van Rees DJ, et al. Neutrophils Kill Antibody-Opsonized Cancer Cells by Trogoptosis. Cell Rep. 2018 Jun 26;23(13):3946-3959.e6.

[0621] 35. Frankel AE, Laver JH, Willingham MC, Burns LJ, Kersey JH, Vallera DA. Therapy of patients with T-cell lymphomas and leukemias using an anti-CD7 monoclonal antibody-ricin A chain immunotoxin. LeukLymphoma. 1997 Jul;26(3-4):287-98.

[0622] 36. DeAngelo DJ, Yu D, Johnson JL, Coutre SE, Stone RM, Stopeck AT, et al. Nelarabine induces complete remissions in adults with relapsed or refractory. Blood. 2007 Jun 15;109(12):5136-42.

[0623] 37. Jain N, Lamb AV, O'Brien S, Ravandi F, Konopleva M, Jabbour E, et al. Early T-cell precursor acute lymphoblastic leukemia / lymphoma (ETP-ALL / LBL) in adolescents and adults: a high-risk subtype. Blood. 2016 Apr 14;127(15):1863-9.

[0624] Glossary

[0625] AML Acute Myeloid Leukemia

[0626] ADCC Antibody-dependent cell-mediated cytotoxicity

[0627] ADCP antibody-dependent cell-mediated phagocytosis

[0628] CAR-T Chimeric Antigen Receptor T Cells

[0629] CDC Complement-dependent cytotoxicity

[0630] CEBPA CCAAT / enhancer binding protein alpha gene

[0631] CRP complement regulatory protein

[0632] DC Development Candidates

[0633] HSC Hematopoietic Stem Cell

[0634] mAb monoclonal antibody

[0635] MOA Action Mode

[0636] MRD minimal residual disease

[0637] PBMC peripheral blood mononuclear cells

[0638] SoC Standards of Care

[0639] Tagg aggregation temperature

[0640] T-ALL T-cell acute lymphoblastic leukemia

[0641] Tm Melting temperature

[0642] T-PLL T-cell prolymphocytic leukemia Glossary

[0643] AML Acute Myeloid Leukemia

[0644] ADCC Antibody-dependent cell-mediated cytotoxicity

[0645] ADCP antibody-dependent cell-mediated phagocytosis

[0646] CAR-T Chimeric Antigen Receptor T Cells

[0647] CDC Complement-dependent cytotoxicity

[0648] CEBPA CCAAT / enhancer binding protein alpha gene

[0649] CRP complement regulatory protein

[0650] DC Development Candidates

[0651] HSC Hematopoietic Stem Cell

[0652] mAb monoclonal antibody

[0653] MOA Action Mode

[0654] MRD minimal residual disease

[0655] PBMC peripheral blood mononuclear cells

[0656] SoC Standards of Care

[0657] Tagg aggregation temperature

[0658] T-ALL T-cell acute lymphoblastic leukemia

[0659] Tm Melting temperature

[0660] T-PLL T-cell prolymphocytic leukemia

[0661] Table 1. Affinity characterization by SPR measurement. Kinetic rates and equilibrium binding constants for SPR of mAbs were obtained by global fitting of sensorgrams using a 1:1 interaction model. nbs = no observed binding.

[0662]

[0663] Table 2. Maximum killing and EC50 values ​​of lead mAbs on CEM cells in CDC assay. from Figure 5 Average maximal killing and EC50 values ​​were obtained from the experiments in .

[0664] MAb Maximum damage <![CDATA[EC 50 ]]> 1730C02 E345R 93.2％ 0.37pM 1734E10 E345R 8.8％ 0.09nM 1734F05 E345R 100.1％ 1.16nM 1738B07 E345R 75.4％ 2.25nM 1741E04 E345R 99.8％ 0.12nM 1741G09 E345R 99.5％ 0.12nM 1896A03 E345R 69.5％ 0.60nM TH69 E345R 87.1％ 0.21nM RFT2 E345R 98.5％ 0.22nM

[0665] Table 3. Maximum killing and EC of 1741G09 E430G against 14 T-ALL cell lines in CDC assay 50 value.These data were obtained from the experiment in Figure 8. The relative expression levels of CD7 are based on the data from Tables 8 and 9.

[0666]

[0667] Table 4. Cytokine levels from PBMCs treated with 1741G09 E430G and control antibodies. IgG1E430G antibody was included as a negative control, and anti-CD3 and anti-CD28 as positive controls. PBMCs from 5 donors were treated with different antibodies at a concentration of 60 μg / ml for 48 hours at 37°C.

[0668]

[0669]

[0670] Table 5: Immunophenotyping of T-ALL / LBL using WHO criteria (37)

[0671]

[0672] Table 6: Statistical comparison of antibodies presented in Figure 6 middle

[0673]

[0674]

[0675] Table 7: Clinical and genetic profiles of PDTALL cell lines

[0676]

[0677] Table 8: CD7 and CRP expression profiles of non-relapsed T-ALL cell lines

[0678]

[0679] Table 9: CD7 and CRP expression profiles of relapsed T-ALL cell lines

[0680]

[0681] Table 10: Biophysical Summary of 1741G09 E430G

[0682]

[0683] Table 11: Sequence

[0684] Sequences for use in the present invention.

[0685]

[0686]

[0687]

[0688]

[0689]

[0690]

[0691]

[0692]

[0693]

[0694]

[0695]

[0696]

[0697]

[0698]

[0699]

[0700]

[0701]

[0702]

[0703]

[0704]

[0705]

[0706]

[0707]

[0708]

[0709]

[0710]

[0711]

[0712]

[0713]

[0714]

[0715]

[0716]

[0717]

[0718]

[0719]

[0720]

[0721]

[0722]

[0723]

[0724] Table 12: Gene segment usage

[0725]

Claims

1. An antibody or fragment that specifically binds to CD7 (cluster of differentiation 7), The antibodies or fragments include: (a) a VH domain comprising a CDRH1 sequence of SEQ ID NO: 1, a CDRH2 sequence of SEQ ID NO: 2, and a CDRH3 sequence of SEQ ID NO: 3; and a VL domain comprising a CDRL1 sequence of SEQ ID NO: 11, a CDRL2 sequence of SEQ ID NO: 12, and a CDRL3 sequence of SEQ ID NO: 13; or (b) a VH domain comprising a CDRH1 sequence of SEQ ID NO: 4, a CDRH2 sequence of SEQ ID NO: 5, and a CDRH3 sequence of SEQ ID NO: 6; and a VL domain comprising a CDRL1 sequence of SEQ ID NO: 14, a CDRL2 sequence of SEQ ID NO: 15, and a CDRL3 sequence of SEQ ID NO:

16.

2. An antibody or fragment that specifically binds to CD7 (cluster of differentiation 7), The antibodies or fragments include: (a) a VH domain comprising a CDRH1 sequence of SEQ ID NO: 61, a CDRH2 sequence of SEQ ID NO: 62, and a CDRH3 sequence of SEQ ID NO: 63; and a VL domain comprising a CDRL1 sequence of SEQ ID NO: 71, a CDRL2 sequence of SEQ ID NO: 72, and a CDRL3 sequence of SEQ ID NO: 73; or (b) a VH domain comprising a CDRH1 sequence of SEQ ID NO: 64, a CDRH2 sequence of SEQ ID NO: 65, and a CDRH3 sequence of SEQ ID NO: 66; and a VL domain comprising a CDRL1 sequence of SEQ ID NO: 74, a CDRL2 sequence of SEQ ID NO: 75, and a CDRL3 sequence of SEQ ID NO:

76.

3. The antibody or fragment according to claim 1 or 2, wherein the antibody or fragment comprises a constant region enabling it to exert effector function.

4. The antibody or fragment according to claim 1, wherein the antibody or fragment comprises (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 7; or an amino acid sequence at least 70% identical thereto; or (b) a VL domain comprising the amino acid sequence of SEQ ID NO: 17; or an amino acid sequence at least 70% identical thereto; or (c) a VH domain comprising SEQ ID NO: 7 and a VL domain comprising SEQ ID NO:

17.

5. The antibody or fragment according to claim 2, wherein the antibody or fragment comprises (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 67; or an amino acid sequence at least 70% identical thereto; or (b) a VL domain comprising the amino acid sequence of SEQ ID NO: 77; or an amino acid sequence at least 70% identical thereto; or (c) a VH domain comprising SEQ ID NO: 67, and a VL domain comprising SEQ ID NO:

77.

6. The antibody or fragment according to any one of claims 1 or 4, comprising: (a) the heavy chain amino acid sequence of SEQ ID NO: 9; or an amino acid sequence at least 70% identical thereto; and / or (b) the light chain amino acid sequence of SEQ ID NO: 19; or an amino acid sequence that is at least 70% identical thereto.

7. The antibody or fragment according to any one of claims 2 or 5, comprising (a) the heavy chain amino acid sequence of SEQ ID NO: 69; or an amino acid sequence at least 70% identical thereto; and / or (b) the light chain amino acid sequence of SEQ ID NO: 79; or an amino acid sequence at least 70% identical thereto.

8. The antibody or fragment of claim 1 or 2, wherein the antibody or fragment comprises a human constant region comprising the amino acid sequence of SEQ ID NO: 88, 90, 92, 94 or 96.

9. The antibody or fragment of claim 1 or 2, further comprising an antigen binding site that specifically binds to another target antigen.

10. The antibody or fragment of claim 9, wherein the target antigen is human CD5, CD14 or CD19.

11. A combination of an amount of an anti-CD7 antibody or fragment and an amount of a chemotherapeutic agent, wherein the antibody or fragment is the antibody or fragment according to any one of claims 1 to 10.

12. The combination of claim 11 comprising multiple doses of the antibody and / or agent.

13. Use of an antibody or fragment according to any one of claims 1 to 10 or a combination according to claim 11 or 12 in the preparation of a medicament for treating leukemia in a human, wherein the antibody, fragment or combination is administered to the human together with an antagonist of human CD5, CD14 or CD19.

14. Use of the antibody, fragment or combination according to claim 13, wherein the leukemia is acute myeloid leukemia, T-ALL, or relapsed leukemia.

15. Use of the antibody, fragment or combination according to claim 14, wherein the relapsed leukemia is relapsed AML or T-ALL.

16. The antibody or fragment according to any one of claims 1 to 10 or the combination according to claim 11 or 12 in the manufacture of a method for treating a human or animal subject with CD7 + Use in medicine for a cell-mediated disease or condition, wherein the antibody, combined fragment is administered to the subject, and wherein CD7 cells are targeted and killed, wherein the disease or condition is leukemia or myelodysplastic syndrome (MDS).

17. Use of the antibody, fragment or combination according to claim 16, wherein the CD7 cells are targeted and killed by ADCP and / or CDC.

18. Use of an antibody or fragment according to any one of claims 1 to 10 or a combination according to claim 11 or 12 in the manufacture of a medicament, wherein the medicament is for administration to a subject to treat or prevent a CD7-mediated disease or condition, wherein the CD7-mediated disease or condition is leukemia or myelodysplastic syndrome (MDS).

19. Use of an antibody, fragment or combination according to claim 18, wherein the CD7-mediated disease or condition is leukemia.

20. Use of the antibody, fragment or combination according to claim 19, wherein the leukemia is T-ALL.

21. Use of the antibody, fragment or combination according to any one of claims 18 to 20, wherein the antibody or fragment is administered to the subject simultaneously or sequentially with chemotherapy or an immune checkpoint inhibitor.

22. A pharmaceutical composition comprising the antibody or fragment according to any one of claims 1 to 10 or the combination according to claim 11 or 12 and a pharmaceutically acceptable excipient, diluent or carrier.

23. A nucleic acid, (a) encoding the VH domain and VL domain of the antibody or fragment according to any one of claims 1 to 10; (b) encodes: (i) a VH domain comprising the amino acid sequence of SEQ ID NO: 7; and (ii) a VL domain comprising the amino acid sequence of SEQ ID NO: 17; (c) encodes: (i) a VH domain comprising the amino acid sequence of SEQ ID NO: 67; and (ii) a VL domain comprising the amino acid sequence of SEQ ID NO: 77; (d) comprising the nucleotide sequence of SEQ ID NO: 10; and the nucleotide sequence of SEQ ID NO: 20; (e) encoding the heavy chain and light chain of the antibody or fragment according to any one of claims 1 to 10; (f) encodes: (i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 7 and (ii) a light chain comprising the amino acid sequence of SEQ ID NO: 17; (g) comprises a nucleotide sequence encoding the heavy chain sequence of SEQ ID NO: 9; and a nucleotide sequence encoding the light chain sequence of SEQ ID NO: 19; or (h) comprises a nucleotide sequence encoding the heavy chain sequence of SEQ ID NO: 69; and a nucleotide sequence encoding the light chain sequence of SEQ ID NO:

79.

24. A vector comprising the nucleic acid according to claim 23.

25. The vector according to claim 24, wherein the vector is a CHO or HEK293 vector.

26. A host cell comprising the nucleic acid according to claim 23 or the vector according to claim 24 or 25.

27. Use of the antibody or fragment of any one of claims 1 to 10 in the manufacture of a kit or diagnostic reagent for diagnosing a CD7-mediated disease or condition in a subject, wherein the CD7-mediated disease or condition is leukemia or myelodysplastic syndrome (MDS).

28. Use of the antibody or fragment according to claim 27, wherein the CD7-mediated disease or condition is leukemia.

29. Use of the antibody or fragment according to claim 27, wherein the leukemia is T-ALL.

30. Use of the antibody or fragment according to any one of claims 1 to 10 in the manufacture of a kit or an assay reagent for detecting CD7-positive cells in a sample in vitro.

31. An in vitro assay for detecting CD7-positive cells in a sample, said assay comprising combining the antibody or fragment according to any one of claims 1 to 10 with an isolated blood or serum sample and determining that cells comprised by said sample are specifically bound by said antibody or fragment, wherein said in vitro assay is for non-diagnostic purposes.

Images