WO2023057595A1

WO2023057595A1 - Humanized hh1 rew

Info

Publication number: WO2023057595A1
Application number: PCT/EP2022/077858
Authority: WO
Inventors: Roman GENERALOV; Veronique Martine Marie PASCAL; Helen HEYDERDAHL; Ada Helena Vilma Repetto LLAMAZARES; Jan Terje ANDERSEN; Stian FOSS; Jostein Dahle
Original assignee: Nordic Nanovector Asa
Priority date: 2021-10-06
Filing date: 2022-10-06
Publication date: 2023-04-13

Abstract

The present disclosure relates to antibodies, antibody fragments and antibody derivates thereof and conjugates thereof and their use in immunotherapy and immunoconjugate therapy, including radioimmunotherapy of cancer with a humanized antibody with a high cytotoxicity as well as various applications of the antibodies.

Description

Humanized HH1 REW

FIELD

BACKGROUND

The present disclosure relates to anti-CD37 molecules, conjugates thereof and use thereof in the treatment of cancers and autoimmune diseases.

Immunotherapy using monoclonal antibodies (mAbs) has been emerging as a safe and selective method for the treatment of cancer and other diseases.

In particular, the role of monoclonal antibodies in therapies that are based on B-cell depletion, e.g. in the treatment of B-cell malignancies, has expanded since the introduction of rituximab, an antibody that is directed against the CD20 antigen on the B-cell surface.

The CD37 antigen is a cell surface antigen that has not been considered as a target for B cell malignancies to the same extent as the B-cell antigen CD20.

CD37, a member of the tetraspanin superfamily, is a heavily glycosylated cell surface molecule with four transmembrane domains and two extracellular loops.

CD37 expression is observed in normal B-cells, non-Hodgkin's lymphoma (NHL), including mantle cell lymphoma (MCL), Burkitts Lymphoma (BL), small lymphocytic lymphoma (SLL) and follicular lymphoma (FL), marginal zone lymphoma (MZL), Diffuse large B-cell lymphoma (DLBCL), lymphoblastic lymphoma (LL), and chronic lymphoid leukemia (CLL).

This expression pattern makes CD37 an attractive target for antibody-mediated cancer therapy.

CD37 was first described in 1986 and characterized by the murine monoclonal antibody MB-1 (Link et al, 1986). CD37 controls both humoral i.e., the aspect of immunity that is mediated by macromolecules found in extracellular fluids such as secreted antibodies, complement proteins, and certain antimicrobial peptides, and cellular immune responses.

CD37-deficiency in mice leads to spontaneous development of B cell lymphoma, and patients with CD37-negative lymphomas have a worse clinical outcome.

Binding of a CD37-specific mAb to cancer cells may trigger various mechanisms of action: after the antibody binds to the extracellular domain of the CD37 antigen, it may activate the complement cascade and lyse the targeted cell.

Also, an anti-CD37 antibody may mediate antibody-dependent cell-mediated cytotoxicity (ADCC) to the target cell, which occurs after the Fc portion of the bound antibody is recognized by appropriate receptors on cytotoxic cells of the immune system.

In addition, the antibody may alter the ability of B-cells to respond to antigen or other stimuli, and the anti-CD37 antibody may initiate programmed cell death (apoptosis).

Anti-CD37 mAb MB-1 was evaluated in two radio-immunotherapy trials in B-NHL patients (B-cell non-Hodgkin's lymphoma; Press et al., 1989; Kaminski et al., 1992).

Others have also disclosed anti-CD37 mABs that show potential (e.g. WO 2009/019312 by Heider et al., W02012/007576 by Stilgenbauer et. al., and WO 2011/092295 by the present inventors) but there is still a long way to go before CD37 is proven the ideal alternative to CD20 for treating B-cell malignancies.

Thus, it has been shown that the CD37 antigen is frequently expressed on tumor cells in several human B-cell malignancies and on mature normal B-lymphocytes and that anti-CD37-based therapy may be a promising approach for treating B cell malignancies.

Although the anti-CD37 antibodies or antibody-like molecules described above (e.g. MB-1 ) have shown anti-tumor efficacy in B-cell malignancies and the potential to target CD37, there is a need for alternate anti-CD37 molecules to improve the therapeutic applicability of anti-CD37 molecules.

Hence, improved anti-CD37 molecules would be advantageous in the pursuit against new treatments against B-cell malignancies. SUMMARY

One or more aspect(s) of the present disclosure relates to an antibody, antibody fragment or antibody derivative thereof, which comprises, a) a heavy chain variable domain (VH) comprising VH-CDR1 , VH-CDR2 and VH-CDR3, and b) a light chain variable domain (VL) comprising VL-CDR1 , VL-CDR2 and VL-CDR3, wherein, c) the heavy chain variable domain (VH) comprises the amino acid sequence of any one of SEQ ID NO: 1 [heavy chain of H02871], wherein, according to SEQ ID NO: 1 , position 2, or position 11 is I or V, position 12 is V or K, position 38 is K or R, position 48 is M or I, position 68 is A or V, position 70 is I or L, position 72 is R or V, position 81 is I or M, and wherein i. the heavy chain VH-CDR1 comprises the amino acid sequence GYSFTD, ii. the heavy chain VH-CDR2 comprises the amino acid sequence PYN, iii. the heavy chain VH-CDR3 comprises the amino acid sequence PYGHYAM, d) the light chain variable domain (VL) comprises the amino acid sequence of any one of SEQ ID NO: 8 [light chain of H02871], wherein, according to SEQ ID NO: 8, position 13 is A or T, position 43 is A or S, position 49 is Y or N, position 71 is F or Y, position 78 is M or L, position 106 is I, M, or V, position 110 is V or D, and wherein i. the light chain VL-CDR1 comprises the amino acid sequence ASQDVST, ii. the light chain VL-CDR2 comprises the amino acid sequence WA, iii. the light chain VL-CDR3 comprises the amino acid sequence HYSTP, e) a lambda or kappa light chain constant domain of human origin and, f) an IgG 1 , lgG2, lgG3 or lgG4, IgM, IgA, IgE or IgD heavy chain constant domain of human origin, wherein the heavy chain constant domain comprises one or more amino acid sequences variants(s), at one or more positions selected from the group consisting of 311 , 434, 428, 438, and 435, with reference to the IgG 1 heavy chain constant domain of SEQ ID NOs: 21 [heavy chain constant domain of lgG1], or synonymous positions in lgG2, lgG3, lgG4, IgM, IgA, IgE or IgD.

In one embodiment, the antibody, antibody fragment or antibody derivative thereof is an anti-CD37 antibody, antibody fragment or antibody derivative thereof.

In another embodiment, the antibody, antibody fragment or antibody derivative thereof is a monoclonal antibody. In a further embodiment, the antibody, antibody fragment or antibody derivative thereof is a fragment selected from the group consisting of a Fab, Fab’, scFV, F(ab’)2, F(ab)2, F(ab)s and scFv- Fc fragment.

In a further embodiment, the antibody, antibody fragment or antibody derivative thereof is a fragment selected from the group consisting of an scFV and scFv-Fc fragment.

In yet another embodiment, the antibody, antibody fragment or antibody derivative thereof the antibody fragment is a minibody, diabody, triabody, or tetrabody.

In yet a further embodiment, the antibody, antibody fragment or antibody derivative thereof have a heavy chain variable domain (VH) that comprises the amino acid sequence of any one of SEQ ID NOs: 1-7 [VH sequence of AH02871 , AH02875, AH02877, AH02879, AH02886 and AH02895] and a light chain variable domain (VL) comprises the amino acid sequence of any one of SEQ ID NOs: 8-18, 87 [VL sequences of AH02871 , AH02875, AH02877, AH02879, AH02886, AH02895, AH02877J106M, AH02877J 106V, AH02877_V110D, AHO2877_I1O6M_V1 10D and

AH02877 I106V V110D],

In a preferred embodiment, the antibody, antibody fragment or antibody derivative thereof have a heavy chain variable domain (VH) that comprises the amino acid of SEQ ID NO: 2 [VH sequence of AH02871] and a light chain variable domain (VL) that comprises the amino acid sequence of any one of SEQ ID NO: 10, 14-18 [VL sequences of AH02877, AH02877J106M, AH02877J 106V, AH02877 V110D, AHO2877_I1O6M_V110D and AH02877J106V V110D],

In a more preferred embodiment, the antibody, antibody fragment or antibody derivative thereof have a heavy chain variable domain (VH) that comprises the amino acid of SEQ ID NO: 2 [VH sequence of AH02871] and a light chain variable domain (VL) that comprises the amino acid sequence of SEQ ID NO: 16 [VL sequences of AH02877 V110D].

In one embodiment, the antibody, antibody fragment or antibody derivative thereof have a predicted immunogenicity risk score (IRS) of the VH domain according to any one of SEQ ID NOs: 1-7 that is lower than the predicted IRS of SEQ ID NO: 19 [VH of Lilotomab].

In another embodiment, the antibody, antibody fragment or antibody derivative thereof have a predicted immunogenicity risk score (IRS) of the VL domain according to any one of SEQ ID NOs: 8-18 that is lower than the predicted IRS of SEQ ID NO: 20 [VL of Lilotomab]. In one embodiment, the antibody, antibody fragment or antibody derivative thereof is an antibody that comprises a lambda or kappa light chain constant domain of human origin and an IgG 1 , lgG2, lgG3 or lgG4, IgM, IgA, IgE or IgD heavy chain constant domain of human origin.

In a further embodiment, the antibody, antibody fragment or antibody derivative thereof is an antibody that comprises a kappa light chain constant domain of human origin and an IgG 1 , lgG2, lgG3 or lgG4 heavy chain constant domain of human origin.

In a preferred embodiment, the antibody, antibody fragment or antibody derivative thereof is an antibody that comprises a kappa light chain constant domain of human origin and an IgG 1 heavy chain constant domain of human origin.

In one or more embodiments, the antibody, antibody fragment or antibody derivative thereof have a heavy chain constant domain that comprises one or more amino acid sequences variants(s), at one or more positions selected from the group consisting of 311 , 434, 428, 438, and 435, with reference to heavy chain constant domain of SEQ ID NOs: 21 [heavy chain constant domain of lgG1],

In a further embodiment said one or more amino acid sequences variants(s), is/are selected from the group consisting of lgG1-Q311 R/N434W/M428E, lgG1-Q31 1 R/N434W, lgG1-Q311 R, lgG1- N434W, lgG3(b)-Q311 R/N434W/M428E, lgG3(b)-Q31 1 R/N434W/Q438E/R435H, lgG1- M252S/Q31 1 R/N434W/M428E, lgG1-Q311 R/N434P/M428E, lgG1-Q311 R/N434W/M428D, lgG1- Q311 R/N434W/M428E/H433K, lgG1-L309K/Q31 1 R/N434W/M428E, lgG1- L309R/Q311 R/N434W/M428E, lgG1-L309S/Q311 R/N434W7M428E, and lgG3(b)- Q311 R/N434W/M428E/R435H.

In a preferred embodiment, the antibody, antibody fragment or antibody derivative thereof is an antibody that comprises a kappa light chain constant domain of human origin and a IgG 1 heavy chain constant domain of human origin, and optionally wherein the IgG 1 heavy chain constant domain further comprises the Q194R/N317W/M31 1 E amino acid sequence variant(s), with reference to SEQ ID NO: 21.

In another preferred embodiment, the antibody, antibody fragment or antibody derivative thereof is an antibody that comprises a kappa light chain constant domain of human origin and a IgG 1 heavy chain constant domain of human origin, and optionally wherein the IgG 1 heavy chain constant domain further comprises the Q311 R/N434W/M428E amino acid sequence variants, according to SEQ ID NO: 22.

In a further preferred embodiment, the heavy chain constant region of said immunoglobulin has an amino acid sequence selected from the group consisting of SEQ ID NOs: 22-34.

Example 9 (table 8 and 9) shows that NNV029 extends the serum half-life of the immunoglobulin as compared to an immunoglobulin lacking said mutation.

Thus, in a further embodiment, said amino acid sequence variant(s) extends the serum half-life of the immunoglobulin as compared to an immunoglobulin lacking said mutation.

In one embodiment, the antibody, antibody fragment or antibody derivative thereof have a heavy chain constant domain of the amino acid sequence according to any one of SEQ ID NOs: 22-34, and wherein the C-terminal residue is K or absent.

In another embodiment, the antibody, antibody fragment or antibody derivative thereof, is an antibody, wherein the C-terminal Lysine in the heavy chain constant domain according to any one of SEQ ID NOs 22-34, is absent and/or removed.

In a preferred embodiment, the amino acid sequence of said antibody, antibody fragment or antibody derivative thereof is a combination of heavy chain and light chain fragments, where said antibody, antibody fragment or antibody derivative comprises, a) a light chain having an amino acid sequence which is SEQ ID NO: 37 and a heavy chain having an amino acid sequence which is SEQ ID NO: 43 [NNV029 heavy chain, AH2871+REW mutations], b) a light chain having an amino acid sequence which is SEQ ID NO: 37 and a heavy chain having an amino acid sequence which is SEQ ID NO: 44 [NNV030 heavy chain, AH2871+REW mutations], or c) a light chain having an amino acid sequence which is SEQ ID NO: 37 and a heavy chain having an amino acid sequence which is SEQ ID NO: 45 [NNV031 heavy chain, AH2871+REW mutations+delCT-Lys].

In one or more embodiments, the antibody, antibody fragment or antibody derivative is glycosylated. In a preferred embodiment, said glycosylation of said antibody, antibody fragment or antibody derivative thereof is fucose deficient.

In a further embodiment said fucose deficient antibody, antibody fragment or antibody derivative thereof have an enhanced and/or improved induction of antibody-dependent cell-mediated cytotoxicity (ADCC), compared to a non-fucose deficient antibody, antibody fragment or antibody derivative thereof.

In one embodiment, the antibody, antibody fragment or antibody derivative thereof is a human or humanized antibody.

In a further embodiment, said human or humanized antibody have an enhanced and/or improved induction of antibody-dependent cell-mediated cytotoxicity (ADCC), antibody dependent cellular phagocytosis (ADCP) and/or complement-dependent cytotoxicity (CDC), compared to a nonhumanized antibody comprising the light chain of SEQ ID NO: 35 [NNV003] and the heavy chain of SEQ ID NO: 36 and/or the therapeutic antibody selected from the group consisting of Rituximab, Obinutuzumab and duohexabody-CD37.

In a further embodiment, said human or humanized antibody have an enhanced and/or improved induction of antibody-dependent cell-mediated cytotoxicity (ADCC), compared to a non-humanized antibody comprising the light chain of SEQ ID NO: 35 [NNV003] and the heavy chain of SEQ ID NO: 36 and/or the therapeutic antibody selected from the group consisting of Rituximab, Obinutuzumab and duohexabody-CD37, optionally in Daudi and/or Ramos cells.

In a yet further embodiment, said human or humanized antibody have an enhanced and/or improved induction of antibody-dependent cell-mediated cytotoxicity (ADCC), antibody dependent cellular phagocytosis (ADCP) and/or complement-dependent cytotoxicity (CDC), compared to a non-humanized antibody comprising the light chain of SEQ ID NO: 35 [NNV003] and the heavy chain of SEQ ID NO: 36 and/or the therapeutic antibody Rituximab.

In a yet further embodiment, said human or humanized antibody have an enhanced and/or improved induction of antibody-dependent cell-mediated cytotoxicity (ADCC), compared to a nonhumanized antibody comprising the light chain of SEQ ID NO: 35 [NNV003] and the heavy chain of SEQ ID NO: 36 and/or the therapeutic antibody Rituximab optionally in Daudi and/or Ramos cells.

In one embodiment, the antibody, antibody fragment or antibody derivative thereof has an affinity for human CD37 expressing cells below 10 nM, such as below 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM and/or such as below 1 nM, such as below 900 pM, 800 pM, 700 pM, 600 pM, 500 pM, 400 pM or 300 pM.

One or more aspect(s) of the present disclosure relates to a nucleic acid sequence encoding an antibody, antibody fragment or antibody derivative thereof according to the present disclosure.

In one embodiment, the nucleic acid sequence encodes an antibody, antibody fragment or antibody derivative thereof that is a combination of heavy chain and light chain fragments, where said antibody, antibody fragment or antibody derivative comprises, a) a light chain having an amino acid sequence which is SEQ ID NO: 37 and a heavy chain having an amino acid sequence which is SEQ ID NO: 43 [NNV029 heavy chain, AH2871+REW mutations], b) a light chain having an amino acid sequence which is SEQ ID NO: 37 and a heavy chain having an amino acid sequence which is SEQ ID NO: 44 [NNV030 heavy chain, AH2871+REW mutations], or c) a light chain having an amino acid sequence which is SEQ ID NO: 37 and a heavy chain having an amino acid sequence which is SEQ ID NO: 45 [NNV031 heavy chain, AH2871+REW mutations+delCT-Lys].

In one embodiment, the nucleic acid sequence encodes an antibody, antibody fragment or antibody derivative thereof with a variable light chain and/or variable heavy chain of any one of SEQ ID NOs: 1-18.

One or more aspect(s) of the present disclosure relates to a nucleic acid construct comprising one or more nucleic acid sequence(s) according to the present disclosure.

One or more aspect(s) of the disclosure relates to a host cell comprising one or more nucleic acid sequence(s) according the present disclosure and/or nucleic acid construct(s) the present disclosure.

In one embodiment, the host cell is a mammalian cell selected from the group consisting of Chinese hamster ovary (CHO) cells, CHO-K1 , CHO-DG44, mouse myeloma (NS0) cells, baby hamster kidney (BHK) cells, and human embryonic kidney lines (HEK293) cells, or an insect cell.

In one embodiment, the host cell is capable of producing an antibody, antibody fragment or antibody derivative thereof according to the present disclosure, wherein the cellular fucose glycosylation pathway of said host cell is modulated, such that the host cell produces a fucose deficient antibody, antibody fragment or antibody derivative thereof.

One or more aspect(s) of the present disclosure relates to an antibody, antibody fragment or antibody derivative thereof according to the present disclosure, produced in a host cell according to the present disclosure.

One or more aspect(s) of the present disclosure relates to an antibody, antibody fragment or antibody derivative thereof, drug conjugate that binds to human CD37 comprising: a) an antibody, antibody fragment or antibody derivative thereof according to the present disclosure, b) a linker, and c) a drug selected from the group consisting of a toxin, a radioisotope, an anticancer drug, a cytotoxic drug and a cytostatic drug.

In one embodiment, said linker is a chelating linker.

In a preferred embodiment, said linker is a chelating linker selected from the group consisting of p- SCN-bn-DOTA, DOTA-NHS-ester and p-SCN-Bn-TCMC.

In one embodiment, said drug is a radionuclide, selected from the group consisting of ²¹¹At, ²¹³Bi, ²¹²Bi, ²¹²Pb, ²²⁵Ac, ²²⁷Th, ⁹⁰Y, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁹⁹Au, ¹⁹⁴lr, ¹⁶⁶Ho, ¹⁵⁹Gd, ¹⁵³Sm, ¹⁶¹Tb, ¹⁴⁹Pm, ¹⁴²Pr, ¹¹¹Ag, ¹⁰⁹Pd, ⁷⁷ As, ⁶⁷Cu, ⁶⁴Cu, ⁴⁷Sc, and ¹⁷⁷Lu.

In another embodiment said drug is an anticancer drug.

One or more aspect(s) of the present disclosure relates to a pharmaceutical composition comprising, as the active ingredient, one or more antibody/antibodies, antibody fragment(s) or antibody derivative(s) thereof and/or an antibody, antibody fragment or antibody derivative thereof drug conjugate according to the present disclosure, and a pharmaceutically acceptable carrier.

In one embodiment, said composition further comprises an additional therapeutic agent, preferably selected in the group consisting of alkylating agents, angiogenesis inhibitors, antibodies, antimetabolites, antimitotics, antiproliferatives, antivirals, aurora kinase inhibitors, apoptosis promoters (for example, Bcl-2 family inhibitors), activators of death receptor pathway, Bcr-Abl kinase inhibitors, BiTE (Bi-Specific T cell Engager) antibodies, antibody drug conjugates, biologic response modifiers, Bruton's tyrosine kinase (BTK) inhibitors, cyclin-dependent kinase inhibitors, cell cycle inhibitors, cyclooxygenase-2 inhibitors, DVDs, leukemia viral oncogene homolog (ErbB2) receptor inhibitors, growth factor inhibitors, heat shock protein (HSP)-90 inhibitors, histone deacetylase (HDAC) inhibitors, hormonal therapies, immunologicals, inhibitors of inhibitors of apoptosis proteins (lAPs), intercalating antibiotics, kinase inhibitors, kinesin inhibitors, Jak2 inhibitors, mammalian target of rapamycin inhibitors, microRNAs, mitogen-activated extracellular signal- regulated kinase inhibitors, multivalent binding proteins, non-steroidal anti-inflammatory drugs (NSAIDs), poly ADP (adenosine diphosphate)-ribose polymerase (PARP) inhibitors, platinum chemotherapeutics, polo-like kinase (Plk) inhibitors, phosphoinositide-3 kinase (PI3K) inhibitors, proteasome inhibitors, purine analogs, pyrimidine analogs, receptor tyrosine kinase inhibitors, retinoids/deltoids plant alkaloids, small inhibitory ribonucleic acids (siRNAs), topoisomerase inhibitors, ubiquitin ligase inhibitors, hypomethylating agents, checkpoints inhibitors, peptide vaccine and the like, epitopes or neoepitopes from tumor antigens, as well as combinations of one or more of these agents.

One or more aspect(s) of the present disclosure relates to a method for producing an antibody, antibody fragment or antibody derivative thereof according to the present disclosure, the method comprising, a) introducing into a mammalian host cell one or more nucleic acid construct(s) of the present disclosure, b) culturing said host cell in a suitable media, c) recovering said antibody, antibody fragment or antibody derivative thereof from the culturing broth, and d) purifying the antibody, antibody fragment or antibody derivative thereof.

In one embodiment of said method, the host cell is a mammalian cell selected from the group consisting of Chinese hamster ovary (CHO) cells, CHO-K1 , CHO-DG44, mouse myeloma (NSO) cells, baby hamster kidney (BHK) cells, and human embryonic kidney lines (HEK293) cells, or an insect cell.

In one embodiment of said method, the cellular fucose glycosylation pathway of said host cell is modulated, such that the host cell produces a fucose deficient antibody, antibody fragment or antibody derivative thereof.

One or more aspect(s) of the present disclosure relates to a method of depleting CD37 expressing B-cells from a population of cells, comprising administering to said population of cells, an antibody, antibody fragment or antibody derivative thereof, an antibody, antibody fragment or antibody derivative thereof, drug conjugate, or a pharmaceutical composition according to the present disclosure.

One or more aspect(s) of the present disclosure relates to a method of treating disease, wherein targeting of CD37 expressing B-cells can provide an inhibition and/or amelioration of said disease, comprising administering a therapeutically effective amount of an antibody, antibody fragment or antibody derivative thereof, an antibody, antibody fragment or antibody derivative thereof, drug conjugate, or a pharmaceutical composition according to the present disclosure.

One or more aspect(s) of the present disclosure relates to a method of treating cancer and/or inflammatory disease(s) and/or autoimmune disease(s) comprising administering a therapeutically effective amount of an antibody, antibody fragment or antibody derivative thereof, and/or a drug conjugate thereof, or a pharmaceutical composition according to the present disclosure.

One or more aspect(s) of the present disclosure relates to a method of treating cancer comprising administering a therapeutically effective amount of an antibody, antibody fragment or antibody derivative thereof, and/or a drug conjugate thereof, or a pharmaceutical composition according to the present disclosure.

One or more aspect(s) the present disclosure relates to the use of an antibody, antibody fragment or antibody derivative thereof, and/ or a drug conjugate thereof, or a pharmaceutical composition according to the present disclosure, in inhibiting cancer and/or inflammatory disease(s) and/or autoimmune diseases.

One or more aspect(s) of the present disclosure relates to the use of an antibody, antibody fragment or antibody derivative thereof, and/or a drug conjugate thereof or a pharmaceutical composition according to the present disclosure, in ameliorating cancer and/or inflammatory disease(s) and/or autoimmune diseases.

One or more aspect(s) of the present disclosure relates to an antibody, antibody fragment or antibody derivative thereof, and/or a drug conjugate thereof, or a pharmaceutical composition according to the present disclosure, for use as a medicament.

In one embodiment said medicament is for use in the treatment of cancer.

In a preferred embodiment said medicament is for use in the treatment of B-cell malignancies. In a more preferred embodiment, said medicament is for treating of a B-cell malignancy selected from the group consisting of B-cell non-Hodgkin’s lymphoma, B-cell chronic lymphocytic leukemia, hairy cell leukemia, lymphoplasmacytic lymphoma and multiple myeloma, comprising administering to the individual in need thereof, an effective amount of an antibody, antibody fragment or antibody derivative thereof, and/or a drug conjugate thereof, or a pharmaceutical composition according to the present disclosure.

In one embodiment said medicament is for treating of inflammatory and autoimmune diseases wherein CD37-positive B cells are enriched.

In one embodiment said medicament is administered once or sequential.

One or more aspect(s) of the present disclosure relates to a formulation of an antibody, antibody fragment or antibody derivative thereof, and/or a drug conjugate thereof, or a pharmaceutical composition according to the present disclosure, for use in pre-treatment, wherein human CD37 is blocked in normal tissues before treatment with immunotoxic anti-CD37 or immunotoxic antibodydrug conjugate.

In one embodiment said formulation is suitable for administration by one or more administration routes selected from the group consisting of oral, topical, intravenous, intramuscular, and subcutaneous administration.

In one embodiment, the amount of the antibody fragment or antibody derivative thereof, and/or a drug conjugate thereof, according to the present disclosure is at least 0.1 mg and not more than 1 g-

One or more aspect(s) of the present disclosure relates to a kit for the production of an antibody fragment or antibody derivative thereof, and/or a drug conjugate thereof according to the present disclosure comprising, a) two or more vials, wherein one vial contains a conjugate comprising a drug linked to a linker, and one vial comprising an antibody fragment or antibody derivative thereof according to the present disclosure, and b) optionally instructions for preparing said antibody-drug conjugate.

One or more aspect(s) of the present disclosure relates A kit for the production of an antibody fragment or antibody derivative thereof, drug conjugate according to the present disclosure comprising, a) two or more vials, wherein one vial contains a conjugate comprising a chelator linked to an antibody fragment or antibody derivative thereof according to the present disclosure, a second vial containing a radionuclide, and b) optionally, instructions for preparing said antibody-radionuclide conjugate.

One or more aspect(s) of the present disclosure relates to an antibody, antibody fragment or antibody derivative thereof, and/or conjugates thereof that binds to human CD37 comprising: a) an antibody, antibody fragment or antibody derivative thereof according to the present disclosure, b) a linker, and c) a compound enriched in one or more isotopes selected from the group consisting of 1¹C, ¹³N, ¹⁵O, ¹⁸F, ⁶⁴Cu and ⁸⁹Zr.

One or more aspect(s) of the present disclosure relates to an antibody, antibody fragment or antibody derivative thereof and/or conjugates thereof according to the present disclosure, for use in positron emission tomography imaging.

In one embodiment, said imaging is for providing diagnosis, staging, and monitoring treatment of cancers.

In another embodiment said cancer is B-cell non-Hodgkin’s lymphoma, B-cell chronic lymphocytic leukemia, hairy cell leukemia, lymphoplasmacytic lymphoma and multiple myeloma

One or more aspect(s) of the present disclosure relates to a pharmaceutical composition, comprising an antibody fragment or antibody derivative thereof, or an antibody fragment or antibody derivative thereof, drug conjugate according to the present disclosure, further comprising one or more further molecule(s), wherein the further molecules is selected from the group consisting of one or more antibodies, small molecule(s), peptide(s) and toxin(s).

BRIEF DESCRIPTION OF FIGURES

Figure 1 [SDS-PAGE]

SDS-PAGE analysis of the antibodies. SDS-PAGE analysis of antibodies under (A) non-reducing and (B) reducing conditions. Figure 2 [Coating levels of the antibodies in ELISA]

Coating levels of the antibodies in ELISA. ELISA showing detection of antibodies (1000 - 0.45 ng/mL) coated in wells using (A-B) either an AP-conjugated anti-human IgG (Fc-specific) antibody or (C-D) an AP-conjugated anti-human kappa light chain antibody. Shown as mean±s.d. of duplicates.

Figure 3 [Binding of the antibodies to FcRn]

Binding of the antibodies to FcRn at acidic and neutral pH. ELISA results showing binding of IgG 1 variants (1000 - 0.45 ng/mL) to biotinylated human FcRn at (A-B) pH 6.0 and (C-D) pH 7.4. Shown as mean s.d. of duplicates.

Figure 4 [FcRn binding kinetics of the antibodies at acidic pH]

FcRn binding kinetics of the antibodies at acidic pH. Sensorgrams showing binding of FcRn-Hisex (1000 - 7.91 nM or 125 - 0.97 nM) to immobilized (A) obinutuzumab, (B) DuoHexabody-CD37, (C) NNV025, (D) NNV023, (E) NNV024, (F) NNV029, (G) NNV030, (H) NNV031 and (I) NNV032 (300 RD) at pH 6.0. The experiments were performed at 25°C with a flow rate of 50 pL/minute.

Figure 5 [SPR binding responses between FcRn]

SPR binding responses between FcRn and the antibodies at pH 7.4. Sensorgrams showing binding of FcRn-Hisex (1000 nM) to immobilized (A) obinutuzumab, (B) DuoHexabody-CD37, (C) NNV025, (D) NNV023, (E) NNV024, (F) NNV029, (G) NNV030, (H) NNV031 and (I) NNV032 (300 RD) at pH 7.4. The experiments were performed at 25°C with a flow rate of 20 pL/minute.

Figure 6 [Binding of the antibodies to low affinity human FcyRs]

Binding of the antibodies to low affinity human FcyRs. ELISA showing binding of the antibodies (10.000 - 4.5 ng/mL) to biotinylated human (A) FcgRllla-V158, (B) FcgRllla-F158, (C) FcgRIIIb, (D) FcgRlla-H131 , (E) FcgRlla-R131 and (F) FcgRIlb. Shown as mean±s.d. of duplicates.

Figure 7 [Binding of the antibodies to human FcgRI.]

Binding of the antibodies to human FcgRI. ELISA showing binding of the antibodies (10.000- 4.5 ng/mL) to biotinylated human FcgRI. Shown as mean ± s.d. of duplicates. Figure 8 [Binding of the antibodies to human C1q]

Binding of the antibodies to human C1q. (A-C) ELISA showing binding of the antibodies (20.000 - 156.25 ng/mL) to human complement factor C1q. Shown as mean ± s.d. of duplicates.

Figure 9 [Activation ofADCC]

Activation of ADCC induced by NNV antibodies, Obinutuzumab (Obin), and duohexabody-CD37 (DXBD37).

Figure 10 [Activation ofADCP via the low affinity FcyRlla-H131 signalling pathway]

Activation of a reporter gene via the low affinity FcyRlla-H131 signalling pathway. The bar plot demonstrates recruitment of the effector cells expressing FcyRlla-H131 receptors in response to different concentrations of the test antibodies.

Figure 11 [Activation ofADCP through the low affinity FcyRlla-R131]

Activation of a reporter gene through the low affinity FcyRlla-R131 signalling pathway. The bar plot demonstrates recruitment of the effector cells expressing FcyRlla-R131 receptors in response to different concentrations of the test antibodies.

Figure 12 [Activation ofADCP through the high affinity FcyRI signalling pathway]

Activation of a reporter gene through the high affinity FcyRI signalling pathway. The bar plot demonstrates recruitment of the effector cells through FcyRI receptors in response to different concentrations of the test antibodies.

Figure 13 [Overview of the ELISA assays]

Overview of the ELISA assays. (A) Illustration of the MicroVue C4d EIA kit. Complement factor C4d in NHS incubated with antibodies are captured by an anti-C4d antibody and visualized by addition of a secondary enzyme-linked anti-C4d antibody. (B) Illustration of the MicroVue CIC EIA kit. Low amounts of human C1q are coated in wells. As efficient binding between C1q and IgG requires high avidity, only IgG complexes/hexamers are bound. Bound IgG complexes/hexamers are visualized by addition of an enzyme-linked anti-human IgG antibody. Figure 14 [Solution-phase complement activation]

Solution-phase complement activation. Measured concentrations of (A) C4d and (B) IgG complexes in NHS incubated for 1 hours at 37°C with 200 mg/ml of the antibodies. Shown as mean±s.d of individual samples analyzed in duplicates. ***p<0.001 by one-way ANOVA test.

Figure 15 [Tm (°C) of the antibodies]

Tm (°C) of the antibodies. Tm (°C) values of the antibodies measured by nanoDSF. Shown as mean ± s.d. of triplicates.

Figure 16 [Aggregation propensity of the antibodies]

Aggregation propensity of the antibodies. Scattering peaks of (A) obinutuzumab, DuoHexabody- CD37, NNV023, NNV025, NNV024 and (B) NNV029, NNV030, NNV031 , NNV032 as a function of temperature (°C). Each line represents the mean of triplicates.

Figure 17 [experimental setup of the HERA assay]

Illustration showing the steps and experimental setup of the HERA assay. 1 : 7.5x104 cells seeded per well in 48-well plates, 1 day prior to the assay. 2: On the day of the assay plates are washed 2x and starved for 1 hour in HBSS before addition of the test antibodies. 3: The plates are inclubated for 3 hours to allow internalization of the test antibodies. The plates are washed before lysate is collected from plate 1 (update fraction). 4: Medium is added to plate 2 and inclubated for 3 h before medium samples are collected (recycled fraction). 5: Plate 2 is washed with ice-cold HBSS, before isolation of cell lysate (residual fraction).

Figure 18 [FcRn mediated handling of the antibodies in HMEC1 cells]

FcRn mediated handling of the antibodies in HMEC1 cells. Figure showing the amounts of antibodies (A) taken up, (B) recycled and (C) accumulated (residual amounts) in HMEC1 cells. Shown as mean ± s.d of triplicates.

Figure 19 [HERA scores]

HERA scores. Figure showing the HERA score for each antibody. The HERA score was determined by dividing the recycled amount of each IgG variant by the intracellular residual amount at the assay endpoint. Shown as mean ± s.d. of triplicates.

SUBSTITUTE SHEET (RULE 26) Figure 20 [CDC effects on Raji cells]

CDC Effects on cell viability induced by NNV Abs, obinutuzumab, and Duohexabody-37 on Raji target cells. A. Clustered bar plot representing CDC effects in presence of 12,5% Human Serum Complement (CTS-006, Creative Biolabs). B. Clustered bar plot representing CDC effects in 12,5% C3&C5 Removed Human Serum (CTS-054, Creative Biolabs). On the x axis, antibody concentration: CTR (0 pg/mL), 0,016, 0,4, 10 pg/mL; on the y axis, relative cell viability, expressed as % of cell viability compared to control cells not treated with any antibody. Results from two independent biological replicates.

Figure 21 [CDC effects on Daudi cells]

CDC Effects on cell viability induced by NNV Abs, obinutuzumab, and Duohexabody-37 on Daudi target cells. A. Clustered bar plot representing CDC effects in presence of 12,5% Human Serum Complement (CTS-006, Creative Biolabs). B. Clustered bar plot representing CDC effects in 12,5% C3&C5 Removed Human Serum (CTS-054, Creative Biolabs). On the x axis, antibody concentration: CTR (0 pg/mL), 0,016, 0,4, 10 pg/mL; on the y axis, relative cell viability, expressed as % of cell viability compared to control cells not treated with any antibody. Results from two independent biological replicates.

Figure 22 [Plasma half life]

Plasma half-life of antibodies in Tg32 hemizygous mice in the absence of competition. Percent antibody remaining in plasma over time. NNV023 (n=5) is shown compared to (A) NNV025 (n=4), (B) NNV024 (n=4), (C) NNV029 (n=5), (D) NNV030 (n=4), (E) NNV031 (n=5), (F) NNV032 (n=5) and (G) obinutuzumab (n=5) and DuoHexabody-CD37 (n=5). Shown as mean s.d. ***p<0.001 , **p<0.002 and ns = not significant by two-way ANOVA.

Figure 23 [Plasma Cone]

Plasma concentration of antibodies in Tg32 hemizygous mice in the absence of competition. Plasma half-life of IgG antibodies shown as percent remaining over time. NNV023 (n=5) is shown compared to (A) NNV025 (n=4), (B) NNV024 (n=4), (C) NNV029 (n=5), (D) NNV030 (n=4), (E) NNV031 (n=5), (F) NNV032 (n=5) and (G) obinutuzumab (n=5) and DuoHexabody-CD37 (n=5). Shown as mean+Z-s.d. ***p<0.001 , *p<0.033 and ns = not significant by two-way ANOVA.

Figure 24 [Establishment of ELISA]

Establishment of an ELISA assay for detection of NNV023 and NNV029 in plasma from IVIg pre- loaded Tg32 hemizygous mice. (A) Illustration showing the ELISA set-up used to specifically capture NNV023 and NNV029 in the presence of excess amounts of IVIg. (B) ELISA results

SUBSTITUTE SHEET (RULE 26) showing binding of titrated amounts (1000.0 - 0. 488 ng/mL) of NNV023 and NNV029 to AbD34091_Fab in the presence of 250 pg/mL IVIg. IVIg only (250 pg/mL and 125 pg/mL) and Blank (PBS/T/S) were included as controls. Shown as mean+Z-s.d of duplicates.

Figure 25 [IgG concentrations]

Total IgG concentrations in plasma from IVIg pre-loaded Tg32 hemizygous mice. Total concentration of human IgG in plasma from IVIg pre-loaded mice administered with NNV023 (n=4) and NNV029 (n=4). Shown as mean s.d. ns = not significant by two-way ANOVA.

Figure 26 [Plasma half-life]

Plasma half-life and concentration of NNV023 and NNV029 in IVIg pre-loaded Tg32 hemizygous mice. (A) Percent of NNV023 (n=4) and NNV029 (n=4) remaining in plasma isolated from IVIg pre- loaded human Tg32 hemizygous mice over time. (B) Concentration of NNV023 (n=4) and NNV029 (n=4) in plasma from IVIg pre-loaded Tg32 hemizygous mice over time. Shown as mean s.d. ***p<0.001 and *p<0.033 by two-way ANOVA.

Figure 27 [Survival curve]

Survival curve of female SCID FcRn-Z- hFcRn (32) Tg mice with i.v. injected Daudi-lymphoma cells (10 million cells) treated with 2.69 mg/kg of NNV029, NNV031 , NNV032, obinutuzumab or recombinant DuoHexabody-CD37 or 100 mL NaCI. The figure describes survival portions of the animals without Daudi cell originated lymphoma up until 130 days from injection of the test items.

Figure 28 [Mean body weights]

Mean body weights including Standard error of the mean (SEM) of SCID FcRn-Z- hFcRn (32) Tg mice (n=8-9) intravenously injected Daudi-lymphoma cells (10 million cells per mouse) on Day 0. The mice were randomized into treatment groups according to body weight from day -1. Treatment was initiated one day after inoculation (Day 1 ). The animals received 6 different treatments according to Table 2. The figure shows weight changes from arrival (day -14) to study end, at day 130 post inoculation.

Figure 29 [ADCP induction in Burkitt’s lymphoma cells FcyRlla-131R],

Relative ADCP induction was assessed in a panel of Burkitt’s lymphoma cell lines using the ADCP FcyRlla-131 R reporter assay (Promega). Bioluminescent signal is obtained through FcyRllaZNFAT- associated luciferase activation in FcyRlla-131 R expressing effector cells. The ADCP reporter assay response is plotted in function of the antibody concentration. The result is the mean of two technical replicates normalized to the untreated within-the-plate control (untreated Target +

SUBSTITUTE SHEET (RULE 26) Effector cells). The error bars are SD. The spline lines are the fit of the data to a sigmoidal 4PL curve.

Figure 30[ADCP induction in DLBCL cells FcyRlla-131R]

Relative ADCP induction was assessed in a panel of Diffused Large B-Cell Lymphoma (DLBCL) cell lines using the ADCP FcyRlla-131R reporter assay (Promega). Bioluminescent signal is obtained through FcyRlla/NFAT-associated luciferase activation in FcyRlla-131R expressing effector cells. The ADCP reporter assay response is plotted in function of the antibody concentration. The result is the mean of two technical replicates normalized to the untreated within- the-plate control (untreated Target + Effector cells). The error bars are SD. The spline lines are the fit of the data to a sigmoidal 4PL curve. Abbreviations:

Figure 31 [ADCP induction in MCL cells FcyRlla-131R]

Relative ADCP induction was assessed in a panel of Mantle Cell Lymphoma (MCL) cell lines using the ADCP FcyRlla-131 R reporter assay (Promega). Bioluminescent signal is obtained through FcyRlla/NFAT-associated luciferase activation in FcyRlla-131 R expressing effector cells. The ADCP reporter assay response is plotted in function of the antibody concentration. The result is the mean of two technical replicates normalized to the untreated within-the-plate control (untreated Target + Effector cells). The error bars are SD. The spline lines are the fit of the data to a sigmoidal 4PL curve.

Figure 32 [AUC of ADCP FcyRlla-131R]

Area under the curve (AUC) for each antibody tested in the B-NHL cell lines (ADCP assay using FcyRlla-131R). Scatter plot including the Area Under the Curve (AUC) values calculated from the graphs in Figure 1. Each dot represents the corresponding AUC value of each cell line. The black line in each group of scatter represents the mean value for each antibody, calculated from all the cell lines in the graph. Abbreviations: DLBCL - Diffused Large B-Cell Lymphoma; MCL - Mantle Cell Lymphoma.

Figure 33 [ADCP induction in Burkitt’s lymphoma cells FcyRlla-131H],

Relative ADCP induction was assessed in a panel of Burkitt’s lymphoma cell lines using the ADCP FcyRlla-131H reporter assay (Promega). Bioluminescent signal is obtained through FcyRlla/NFAT- associated luciferase activation in FcyRlla-131H expressing effector cells. The ADCP reporter assay response is plotted in function of the antibody concentration. The result is the mean of two

SUBSTITUTE SHEET (RULE 26) technical replicates normalized to the untreated within-the-plate control (untreated Target +

Effector cells). The error bars are SD. The spline lines are the fit of the data to a sigmoidal 4PL curve.

Figure 34 [ADCP induction in DLBCL cells FcyRlla-131H]

Relative ADCP induction was assessed in a panel of Diffused Large B-Cell Lymphoma (DLBCL) cell lines using the ADCP FcyRlla-131H reporter assay (Promega). Bioluminescent signal is obtained through FcyRlla/NFAT-associated luciferase activation in FcyRlla-131H expressing effector cells. The ADCP reporter assay response is plotted in function of the antibody concentration. The result is the mean of two technical replicates normalized to the untreated within- the-plate control (untreated Target + Effector cells). The error bars are SD. The spline lines are the fit of the data to a sigmoidal 4PL curve. Abbreviations:

Figure 35 [ADCP induction in MCL cells FcyRlla-131H]

Relative ADCP induction was assessed in a panel of Mantle Cell Lymphoma (MCL) cell lines using the ADCP FcyRlla-131 H reporter assay (Promega). Bioluminescent signal is obtained through FcyRlla/NFAT-associated luciferase activation in FcyRlla-131 H expressing effector cells. The ADCP reporter assay response is plotted in function of the antibody concentration. The result is the mean of two technical replicates normalized to the untreated within-the-plate control (untreated Target + Effector cells). The error bars are SD. The spline lines are the fit of the data to a sigmoidal 4PL curve.

Figure 36 [AUC of ADCP FcyRlla-131H]

Area under the curve (AUC) for each antibody tested in the B-NHL cell lines (ADCP assay using FcyRlla-131H). Scatter plot including the Area Under the Curve (AUC) values calculated from the graphs in Figure 1. Each dot represents the corresponding AUC value of each cell line. The black line in each group of scatter represents the mean value for each antibody, calculated from all the cell lines in the graph. Abbreviations: DLBCL - Diffused Large B-Cell Lymphoma; MCL - Mantle Cell Lymphoma.

Figure 37 [ADCC induction in Burkitt’s lymphoma cell lines]

Normalized ADCC induction assessed in Burkitt’s lymphoma cell lines. Relative ADCC induction was assessed in a panel of Burkitt’s lymphoma cell lines with ADCC FcyRllla-158V reporter assay (Promega). Bioluminescent signal is obtained through FcyRllla/NFAT-associated luciferase

SUBSTITUTE SHEET (RULE 26) activation in FcyRllla-158V expressing effector cells. The ADCC reporter assay response is plotted in function of the antibody concentration. The result is the mean of two technical replicates normalized to the untreated within-the-plate control (untreated Target + Effector cells). The error bars are SD. The spline lines are the fit of the data to a sigmoidal 4PL curve.

Figure 38 [ADCC in DLBCL cell lines]

Normalized ADCC induction assessed in Diffused Large B-Cell Lymphoma cell lines. Relative ADCC induction was assessed in a panel of Diffused Large B-Cell Lymphoma cell lines with ADCC FcyRllla-158V reporter assay (Promega). Bioluminescent signal is obtained through

FcyRllla/NFAT-associated luciferase activation in FcyRllla-158V expressing effector cells. The ADCC reporter assay response is plotted in function of the antibody concentration. The result is the mean of two technical replicates normalized to the untreated within-the-plate control (untreated Target + Effector cells). The error bars are SD. The spline lines are the fit of the data to a sigmoidal 4PL curve.

Figure 39 [ADCC in MCL and ALL cell lines]

Normalized ADCC induction assessed in Mantle Cell Lymphoma (MCL) and Acute Lymphoblastic Leukemia (ALL) cell lines. Relative ADCC induction was assessed in a panel of Diffused Large B- Cell Lymphoma cell lines with ADCC FcyRllla-158V reporter assay (Promega). Bioluminescent signal is obtained through FcyRllla/NFAT-associated luciferase activation in FcyRllla-158V expressing effector cells. The ADCC reporter assay response is plotted in function of the antibody concentration. The result is the mean of two technical replicates normalized to the untreated within- the-plate control (untreated Target + Effector cells). The error bars are SD. The spline lines are the fit of the data to a sigmoidal 4PL curve.

Figure 40 [ADCC parameters]

Main parameters (Emax, EC50, and ADC) for the ADCC Ab concentration-response curves for each antibody tested in the B-NHL cell lines. A. Efficacy (Emax) describes the response range. This parameter expresses a difference between the upper and lower asymptote of a conc.-resp. curve. The parameter is a function of the receptor occupancy and the ability to induce ADCC. The higher the Emax value, the stronger effect achieved at any equal EC50. B. Half Maximal Effective Concentration (EC50) is the potency of a drug. The lower EC50 value, the better potency is. C. Area Under the Curve (AUC) is a function of Emax and EC50 combined. The black line in each group of scatter represents the mean value for each antibody, calculated from all the cell lines in the graph. The results for REH cell line are not shown since the cell line does not express CD20 and CD37 to

SUBSTITUTE SHEET (RULE 26) a sufficient level to generate a concentration-response curve. Abbreviations: DLBCL - Diffused Large B-Cell Lymphoma; MCL - Mantle Cell Lymphoma

DETAILED DESCRIPTION

The present disclosure relates to humanized antibodies, antibody fragments or antibody derivatives thereof from the mouse monoclonal antibody HH1 (lilotomab) and the chimeric monoclonal antibody chHH1 (NNV003).

The antibodies, antibody fragments or antibody derivatives thereof of the present disclosure further comprises one or more amino acid sequences variant(s) that enhances the half-life and Fc binding capacity of the antibodies, antibody fragments or antibody derivatives thereof. Such amino acid sequence variants are in example referred to to as REW substitutions.

An antibody, antibody fragment or antibody derivative thereof

Humanized antibodies, antibody fragments or antibody derivatives thereof are antibodies from nonhuman species whose protein sequences have been modified to increase their similarity to antibody variants produced naturally in humans.

The process of "humanization" is usually applied to monoclonal antibodies developed for administration to humans.

Humanization can be necessary when the process of developing a specific antibody involves generation in a non-human immune system (such as that in mice).

The protein sequences of antibodies produced in this way are partially distinct from homologous antibodies occurring naturally in humans and are therefore, potentially immunogenic when administered to human patients.

Not all monoclonal antibodies designed for human administration need be humanized since many therapies are short-term interventions.

Humanization is usually seen as a distinct from the creation of a mouse-human antibody chimera, such as but not limited to chHH1.

SUBSTITUTE SHEET (RULE 26) So, although the creation of an antibody chimera is normally undertaken to achieve a more humanlike antibody (by substituting the mouse Fc region of the antibody with that from human) simple chimeras of this type are not usually referred to as humanized.

Rather, the protein sequence of a humanized antibody is essentially identical to that of a human variant, despite the non-human origin of some of its complementarity determining region (CDR) segments responsible for the ability of the antibody to bind to its target antigen.

The immunoglobulin heavy chain (Ig-HC) is the large polypeptide subunit of an antibody (immunoglobulin).

A typical antibody is composed of two immunoglobulin (Ig) heavy chains and two Ig light chains.

Several different types of heavy chain exist that define the class or isotype of an antibody.

These heavy chain types vary between different animals.

The immunoglobulin light chain is the small polypeptide subunit of an antibody (immunoglobulin).

There are two types of light chain in humans (as in other mammals), kappa (K) chain, encoded by the immunoglobulin kappa locus on chromosome 2 and the lambda (A) chain, encoded by the immunoglobulin lambda locus on chromosome 22.

Antibodies are produced by B lymphocytes, each expressing only one class of light chain.

Once set, light chain class remains fixed for the life of the B lymphocyte.

In a healthy individual, the total kappa to lambda ratio is roughly 2:1 in serum (measuring intact whole antibodies) or 1 :1 .5 if measuring free light chains, with a highly divergent ratio indicative of neoplasm.

The exact normal ratio of kappa to lambda ranges from 0.26 to 1 .65.

Both the kappa and the lambda chains can increase proportionately, maintaining a normal ratio.

SUBSTITUTE SHEET (RULE 26) Both variable and constant domains in a humanized antibody fragments or antibody derivatives thereof derived from the mouse monoclonal antibody HH1 and/or the chimeric chHH1 can differ from known sequences.

Examples of such variants are clear from the present disclosure and include selection of constant domains, genetic variation of variable chains and variants of the Fc domain in order to modulate effector functions.

The present inventors have genetically engineered, humanized antibody fragments or antibody derivatives thereof, derived from the mouse monoclonal antibody HH1 , lilotomab (NNV001 ) or the chimeric monoclonal antibody chHH1 (NNV003).

These antibodies show a promising effect in the search for optimal treatment of B-cell related malignancies and/or inflammatory disease(s) and/or autoimmune disease(s).

These antibodies show a promising effect in the search for optimal treatment of B-cell related malignancies.

These effects are shown in the experiments of the present disclosure.

Thus, one or more aspect(s) of the present disclosure relates to an antibody, antibody fragment or antibody derivative thereof, which comprises, a heavy chain variable domain (VH) comprising VH-CDR1 , VH-CDR2 and VH-CDR3, a light chain variable domain (VL) comprising VL-CDR1 , VL-CDR2 and VL-CDR3, a lambda or kappa light chain constant domain of human origin, and an IgG 1 , lgG2, lgG3 or lgG4, IgM, IgA, IgE or IgD heavy chain constant domain of human origin, wherein the heavy chain constant domain comprises one or more amino acid sequences variants(s), at one or more positions selected from the group consisting of 311 , 434, 428, 438, and 435, with reference to the IgG 1 heavy chain constant domain of SEQ ID NOs: 21 [heavy chain constant domain of IgG 1 ], or synonymous positions in lgG2, lgG3, lgG4, IgM, IgA, IgE or IgD.

In one or more further aspect(s) of the present disclosure relates to an antibody, antibody fragment or antibody derivative thereof, which comprises, a) a heavy chain variable domain (VH) comprising VH-CDR1 , VH-CDR2 and VH-CDR3, and

SUBSTITUTE SHEET (RULE 26) b) a light chain variable domain (VL) comprising VL-CDR1 , VL-CDR2 and VL-CDR3, wherein, c) the heavy chain variable domain (VH) comprises the amino acid sequence of any one of SEQ ID NO: 1 [heavy chain of H02871], wherein, according to SEQ ID NO: 1 , position 2, or position 11 is I or V, position 12 is V or K, position 38 is K or R, position 48 is M or I, position 68 is A or V, position 70 is I or L, position 72 is R or V, position 81 is I or M, and wherein i. the heavy chain VH-CDR1 comprises the amino acid sequence GYSFTD, ii. the heavy chain VH-CDR2 comprises the amino acid sequence PYN,

Hi. the heavy chain VH-CDR3 comprises the amino acid sequence PYGHYAM, d) the light chain variable domain (VL) comprises the amino acid sequence of any one of SEQ ID NO: 8 [light chain of H02871], wherein, according to SEQ ID NO: 8, position 13 is A or T, position 43 is A or S, position 49 is Y or N, position 71 is F or Y, position 78 is M or L, position 106 is I, M, or V, position 110 is V or D, i. the light chain VL-CDR1 comprises the amino acid sequence ASQDVST, ii. the light chain VL-CDR2 comprises the amino acid sequence WA,

Hi. the light chain VL-CDR3 comprises the amino acid sequence HYSTP, e) a lambda or kappa light chain constant domain of human origin and, f) an IgG 1 , lgG2, lgG3 or lgG4, IgM, IgA, IgE or IgD heavy chain constant domain of human origin, wherein the heavy chain constant domain comprises one or more amino acid sequences variants(s), at one or more positions selected from the group consisting of 311 , 434, 428, 438, and 435, with reference to the IgG 1 heavy chain constant domain of SEQ ID NOs: 21 [heavy chain constant domain of lgG1], or synonymous positions in lgG2, lgG3, lgG4, IgM, IgA, IgE or IgD.

In one or more embodiment(s) the heavy chain variable domain (VH) comprises the amino acid sequence of any one of SEQ ID NO: 1 , or a functional homologue thereof having an amino acid sequence which is at least 80 % identical, such as at least 81 %, 82 %, 83 %, 84 %, 85 %, 86 %, 87 %, 88 %, 89 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 95 %, 96 %, 97 %, 98 %, 99 % or 99.9 % identical to any of the variants of SEQ ID NO: 1 .

In one or more embodiment(s) the heavy chain variable domain (VH) comprises the amino acid sequence of any one of SEQ ID NO: 2, or a functional homologue thereof having an amino acid sequence which is at least 80 % identical, such as at least 81 %, 82 %, 83 %, 84 %, 85 %, 86 %, 87 %, 88 %, 89 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 95 %, 96 %, 97 %, 98 %, 99 % or 99.9 % identical to any of the variants of SEQ ID NO: 2.

SUBSTITUTE SHEET (RULE 26) A functional homologue of an amino acid/nucleic acid sequence as described herein is a amino acid/nucleic acid sequence with alterations in the sequence, which retain its original functionality. A functional homologue may be obtained by mutagenesis. The functional homologue should have a remaining functionality of at least 70%, such as 80 %, 90% or 100% compared to the functionality of the amino acid/nucleic acid sequence.

A functional homologue of any one of the disclosed amino acid or nucleic acid sequences can also have a higher functionality. A functional homologue of any one of the proposed antibodies, antibody fragments or antibody derivates thereof, comprising any one or more of SEQ ID NOs: 1- 18, should ideally retain a high affinity binding to a human CD37 protein, and may induce antibodydependent cell-mediated cytotoxicity (ADCC) in Ramos or Daudi cells or other beneficial effectors according to the present disclosure, furthermore a reduction in consumables, resulting in a lowered production cost or a prolonged shelf life is also a favourable feature.

In one or more exemplified embodiment(s) the heavy chain variable domain (VH) comprises the amino acid sequence of any one of SEQ ID NO: 1.

In one or more embodiment(s), the light chain variable domain (VL) comprises the amino acid sequence of any one of SEQ ID NOs: 8 [light chain of H02871], or a functional homologue thereof having an amino acid sequence which is at least 80 % identical, such as at least 81 %, 82 %, 83 %, 84 %, 85 %, 86 %, 87 %, 88 %, 89 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 95 %, 96 %, 97 %, 98 %, 99 % or 99.9 % identical to any of the variants of SEQ ID NO: 8.

In one or more embodiment(s), the light chain variable domain (VL) comprises the amino acid sequence of SEQ ID NO: 87 [light chain of H02871], or a functional homologue thereof having an amino acid sequence which is at least 80 % identical, such as at least 81 %, 82 %, 83 %, 84 %, 85 %, 86 %, 87 %, 88 %, 89 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 95 %, 96 %, 97 %, 98 %, 99 % or 99.9 % identical to SEQ ID NO: 87.

The term “sequence identity of [a certain] %” in the context of two or more nucleic acid or amino acid sequences means that the two or more sequences have nucleic acids or amino acid residues in common in the given percent, when compared and aligned for maximum correspondence over a comparison window or designated sequences of nucleic acids or amino acids (e.g., the sequences have at least 90 percent (%) identity). Percent identity of nucleic acid or amino acid sequences can be measured using a BLAST 2.0 sequence comparison algorithm with default parameters, or by manual alignment and visual inspection (see e.g. http://www.ncbi.nlm.nih.gov/BLAST/). This definition also applies to the complement of a test sequence and to sequences that have deletions

SUBSTITUTE SHEET (RULE 26) and/or additions, as well as those that have substitutions. An example of an algorithm that is suitable for determining percent identity, sequence similarity and for alignment is the BLAST 2.2.20+ algorithm, which is described in Altschul et al. Nucl. Acids Res. 25, 3389 (1997). BLAST 2.2.20+ is used to determine percent sequence identity for the nucleic acids and proteins of the disclosure. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). Examples of commonly used sequence alignment algorithms are

CLUSTAL Omega (http://www.ebi.ac.uk/Tools/msa/clustalo/), EMBOSS Needle (http://www.ebi.ac.uk/Tools/psa/emboss_needle/), MAFFT (http://mafft.cbrc.jp/alignment/server/), or MUSCLE (http://www.ebi.ac.uk/Tools/msa/muscle/).

In that regard, in one or more embodiment(s) the sequence identity of a sequence is at least 80 % identical compared to a reference sequence. In one or more embodiment(s) the sequence identity of a sequence is at least 81 % identical compared to a reference sequence. In one or more embodiment(s) the sequence identity of a sequence is at least 82 % identical compared to a reference sequence. In one or more embodiment(s) the sequence identity of a sequence is at least 83 % identical compared to a reference sequence. In one or more embodiment(s) the sequence identity of a sequence is at least 84 % identical compared to a reference sequence. In one or more embodiment(s) the sequence identity of a sequence is at least 85 % identical compared to a reference sequence. In one or more embodiment(s) the sequence identity of a sequence is at least 86 % identical compared to a reference sequence. In one or more embodiment(s) the sequence identity of a sequence is at least 87 % identical compared to a reference sequence. In one or more embodiment(s) the sequence identity of a sequence is at least 88 % identical compared to a reference sequence. In one or more embodiment(s) the sequence identity of a sequence is at least 89 % identical compared to a reference sequence. In one or more embodiment(s) the sequence identity of a sequence is at least 90 % identical compared to a reference sequence. In one or more embodiment(s) the sequence identity of a sequence is at least 91 % identical compared to a reference sequence. In one or more embodiment(s) the sequence identity of a sequence is at least 92 % identical compared to a reference sequence. In one or more embodiment(s) the sequence identity of a sequence is at least 93 % identical compared to a reference sequence. In one or more embodiment(s) the sequence identity of a sequence is at least 94 % identical compared to a reference sequence. In one or more embodiment(s) the sequence identity of a sequence is at least 95 % identical compared to a reference sequence. In one or more embodiment(s) the sequence identity of a sequence is at least 96 % identical compared to a reference sequence. In one or more embodiment(s) the sequence identity of a sequence is at least 97 % identical compared to a

SUBSTITUTE SHEET (RULE 26) reference sequence. In one or more embodiment(s) the sequence identity of a sequence is at least 98 % identical compared to a reference sequence. In one or more embodiment(s) the sequence identity of a sequence is at least 99 % identical compared to a reference sequence. In one or more embodiment(s) the sequence identity of a sequence is at least 99,9 % identical compared to a reference sequence.

In one or more embodiment(s) the sequence identity of a sequence is 100 % identical to a reference sequence.

In one or more embodiment(s), the heavy chain variable domain (VH) comprises any one of the amino acid sequence variants of SEQ ID NO: 1 [heavy chain of H02871 w variants] and the light chain variable domain (VL) comprises any one of the amino acid sequence variants of SEQ ID NO: 8 [light chain of H02871 w variants].

In one or more embodiment(s), the heavy chain variable domain (VH) comprises the amino acid sequence of SEQ ID NO: 2 [heavy chain of H02871] and the light chain variable domain (VL) comprises the amino acid sequence of SEQ ID NO: 87 [light chain of H02871].

In one or more embodiments the antibody, antibody fragment or antibody derivative thereof, comprises, a) a heavy chain variable domain (VH) comprising VH-CDR1 , VH-CDR2 and VH-CDR3, and b) a light chain variable domain (VL) comprising VL-CDR1 , VL-CDR2 and VL-CDR3, wherein, c) the heavy chain variable domain (VH) comprises the amino acid sequence of any one of SEQ ID NO: 1 [heavy chain of H02871], wherein, according to SEQ ID NO: 1 , position 2, or position 11 is I or V, position 12 is V or K, position 38 is K or R, position 48 is M or I, position 68 is A or V, position 70 is I or L, position 72 is R or V, position 81 is I or M, and wherein the heavy chain optionally comprises, i. VH-CDR1 of the amino acid sequence GYSFTD, and/or ii. VH-CDR2 of the amino acid sequence PYN, and/or iii. VH-CDR3 of the amino acid sequence PYGHYAM, d) the light chain variable domain (VL) comprises the amino acid sequence of any one of SEQ ID NOs: 8 [light chain of H02871],

SUBSTITUTE SHEET (RULE 26) wherein, according to SEQ ID NO: 8, position 13 is A or T, position 43 is A or S, position 49 is Y or N, position 71 is F or Y, position 78 is M or L, position 106 is I, M, or V, position 110 is V or D and wherein the heavy chain optionally comprises, i. VL-CDR1 of the amino acid sequence ASQDVST and/or, ii. VL-CDR2 of the amino acid sequence WA, iii. VL-CDR3 of the amino acid sequence HYSTP, e) a lambda or kappa light chain constant domain of human origin and, f) an IgG 1 , lgG2, lgG3 or lgG4, IgM, IgA, IgE or IgD heavy chain constant domain of human origin, wherein the heavy chain constant domain comprises one or more amino acid sequences variants(s), at one or more positions selected from the group consisting of 311 , 434, 428, 438, and 435, with reference to the IgG 1 heavy chain constant domain of SEQ ID NOs: 21 [heavy chain constant domain of lgG1], or synonymous positions in lgG2, lgG3, lgG4, IgM, IgA, IgE or IgD.

A light chain variable domain (VL) and/or a heavy chain variable domain (VH)

An antibody, antibody fragment or antibody derivative thereof, may comprise a light chain variable domain (VL) and/or a heavy chain variable domain (VH). In some cases, an antibody, antibody fragment or antibody derivative thereof comprises a light chain variable domain (VL) and no heavy chain variable domain (VH). In other cases, an antibody, antibody fragment or antibody derivative thereof comprises a heavy chain variable domain (VH) and no light chain variable domain (VL)

Thus, in one or more embodiment(s), the antibody, antibody fragment or antibody derivative thereof, comprises a light chain variable domain (VL) and/or a heavy chain variable domain (VH).

VH and VL variants

The different VH and VL variants were developed, in brief, by grafting of the CDRs of lilotomab into human acceptors to obtain a series of humanized light chains and a number of heavy chains for each antibody.

Thus, in one or more exemplified embodiment(s), the antibody, antibody fragment or antibody derivative thereof have a heavy chain variable domain (VH) that comprises the amino acid sequence of any one of SEQ ID NOs: 1-7 [VH sequence of AH02871 , AH02875, AH02877, AH02879, AH02886 and AH02895] and a light chain variable domain (VL) comprises the amino acid sequence of any one of SEQ ID NOs: 8-18 [VL sequences of AH02871 , AH02875, AH02877,

SUBSTITUTE SHEET (RULE 26) AH02879, AH02886, AH02895, AH02877J106M, AH02877J 106V, AH02877_V110D, AH02877J106M V110D and AH02877J106V V110D],

In a preferred embodiment, the antibody, antibody fragment or antibody derivative thereof have a heavy chain variable domain (VH) that comprises the amino acid of SEQ ID NO: 2 [VH sequence of AH02871] and a light chain variable domain (VL) that comprises the amino acid sequence of any one of SEQ ID NOs: 10, 14-18 [VL sequences of AH02877, AH02877J106M, AH02877J106V, AH02877 V110D, AHO2877_I1O6M_V110D and AH02877J106V V110D],

In one or more exemplified embodiment(s), the heavy chain variable domain (VH) comprises the amino acid sequence of SEQ ID NO: 2 [AH02871_HC_VH] and the light chain variable domain (VL) comprises the amino acid sequence of SEQ ID NO: 87 [AH02871 LC VL].

In one or more exemplified embodiment(s), the heavy chain variable domain (VH) comprises the amino acid sequence of SEQ ID NO: 2 [AH02871_HC_VH] and the light chain variable domain (VL) comprises the amino acid sequence of SEQ ID NO: 9 [AH02875 LC VL].

In one or more exemplified embodiment(s), the heavy chain variable domain (VH) comprises the amino acid sequence of SEQ ID NO: 2 [AH02871_HC_VH] and the light chain variable domain (VL) comprises the amino acid sequence of SEQ ID NO: 10 [H02877_LC_VL].

In one or more exemplified embodiment(s), the heavy chain variable domain (VH) comprises the amino acid sequence of SEQ ID NO: 2 [AH02871_HC_VH] and the light chain variable domain (VL) comprises the amino acid sequence of SEQ ID NO: 11 [H02879_LC_VL].

In one or more exemplified embodiment(s), the heavy chain variable domain (VH) comprises the amino acid sequence of SEQ ID NO: 2 [AH02871_HC_VH] and the light chain variable domain (VL) comprises the amino acid sequence of SEQ ID NO: 12 [H02886 LC VL].

In one or more exemplified embodiment(s), the heavy chain variable domain (VH) comprises the amino acid sequence of SEQ ID NO: 2 [AH02871_HC_VH] and the light chain variable domain (VL) comprises the amino acid sequence of SEQ ID NO: 13 [H02895_LC_VL].

In one or more embodiment(s), the heavy chain variable domain (VH) comprises the amino acid sequence of SEQ ID NO: 2 [AH02871_HC_VH] and the light chain variable domain (VL) comprises the amino acid sequence of SEQ ID NO: 14 [H02877 I106M VL].

SUBSTITUTE SHEET (RULE 26) In one or more embodiment(s), the heavy chain variable domain (VH) comprises the amino acid sequence of SEQ ID NO: 2 [AH02871_HC_VH] and the light chain variable domain (VL) comprises the amino acid sequence of SEQ ID NO: 15 [AH02877 I106V VL].

In one or more embodiment(s), the heavy chain variable domain (VH) comprises the amino acid sequence of SEQ ID NO: 2 [AH02871_HC_VH] and the light chain variable domain (VL) comprises the amino acid sequence of SEQ ID NO: 17 [AHO2877_I1O6M_V11OD_VL],

In one or more embodiment(s), the heavy chain variable domain (VH) comprises the amino acid sequence of SEQ ID NO: 2 [AH02871_HC_VH] and the light chain variable domain (VL) comprises the amino acid sequence of SEQ ID NO: 18 [AHO2877_I1O6V_V11OD_VL],

In one or more exemplified embodiment(s), the heavy chain variable domain (VH) comprises the amino acid sequence of SEQ ID NO: 3 [AH02875_HC_VH] and the light chain variable domain (VL) comprises the amino acid sequence of SEQ ID NO: 87 [AH02871 LC VL],

In one or more exemplified embodiment(s), the heavy chain variable domain (VH) comprises the amino acid sequence of SEQ ID NO: 3 [AH02875_HC_VH] and the light chain variable domain (VL) comprises the amino acid sequence of SEQ ID NO: 9 [AH02875 LC VL].

In one or more exemplified embodiment(s), the heavy chain variable domain (VH) comprises the amino acid sequence of SEQ ID NO: 3 [AH02875_HC_VH] and the light chain variable domain (VL) comprises the amino acid sequence of SEQ ID NO: 10 [H02877_LC_VL].

In one or more exemplified embodiment(s), the heavy chain variable domain (VH) comprises the amino acid sequence of SEQ ID NO: 3 [AH02875_HC_VH] and the light chain variable domain (VL) comprises the amino acid sequence of SEQ ID NO: 11 [H02879_LC_VL].

In one or more exemplified embodiment(s), the heavy chain variable domain (VH) comprises the amino acid sequence of SEQ ID NO: 3 [AH02875_HC_VH] and the light chain variable domain (VL) comprises the amino acid sequence of SEQ ID NO: 12 [H02886 LC VL],

In one or more exemplified embodiment(s), the heavy chain variable domain (VH) comprises the amino acid sequence of SEQ ID NO: 3 [AH02875_HC_VH] and the light chain variable domain (VL) comprises the amino acid sequence of SEQ ID NO: 13 [H02895_LC_VL].

SUBSTITUTE SHEET (RULE 26) In one or more embodiment(s), the heavy chain variable domain (VH) comprises the amino acid sequence of SEQ ID NO: 3 [AH02875_HC_VH] and the light chain variable domain (VL) comprises the amino acid sequence of SEQ ID NO: 14 [H02877 I106M VL].

In one or more embodiment(s), the heavy chain variable domain (VH) comprises the amino acid sequence of SEQ ID NO: 3 [AH02875_HC_VH] and the light chain variable domain (VL) comprises the amino acid sequence of SEQ ID NO: 15[AH02877_l106V_VL].

In one or more embodiment(s), the heavy chain variable domain (VH) comprises the amino acid sequence of SEQ ID NO: 3 [AH02875_HC_VH] and the light chain variable domain (VL) comprises the amino acid sequence of SEQ ID NO: 17 [AHO2877_I1O6M_V11OD_VL],

In one or more embodiment(s), the heavy chain variable domain (VH) comprises the amino acid sequence of SEQ ID NO: 3 [AH02875_HC_VH] and the light chain variable domain (VL) comprises the amino acid sequence of SEQ ID NO: 18 [AHO2877_I1O6V_V11OD_VL],

In one or more exemplified embodiment(s), the heavy chain variable domain (VH) comprises the amino acid sequence of SEQ ID NO: 4 [AH02877_HC_VH] and the light chain variable domain (VL) comprises the amino acid sequence of SEQ ID NO: 87 [AH02871 LC VL],

In one or more exemplified embodiment(s), the heavy chain variable domain (VH) comprises the amino acid sequence of SEQ ID NO: 4 [AH02877_HC_VH] and the light chain variable domain (VL) comprises the amino acid sequence of SEQ ID NO: 9 [AH02875 LC VL].

In one or more exemplified embodiment(s), the heavy chain variable domain (VH) comprises the amino acid sequence of SEQ ID NO: 4 [AH02877_HC_VH] and the light chain variable domain (VL) comprises the amino acid sequence of SEQ ID NO: 10 [H02877_LC_VL].

In one or more exemplified embodiment(s), the heavy chain variable domain (VH) comprises the amino acid sequence of SEQ ID NO: 4 [AH02877_HC_VH] and the light chain variable domain (VL) comprises the amino acid sequence of SEQ ID NO: 11 [H02879_LC_VL].

In one or more exemplified embodiment(s), the heavy chain variable domain (VH) comprises the amino acid sequence of SEQ ID NO: 4 [AH02877_HC_VH] and the light chain variable domain (VL) comprises the amino acid sequence of SEQ ID NO: 12 [H02886 LC VL],

SUBSTITUTE SHEET (RULE 26) In one or more exemplified embodiment(s), the heavy chain variable domain (VH) comprises the amino acid sequence of SEQ ID NO: 4 [AH02877_HC_VH] and the light chain variable domain (VL) comprises the amino acid sequence of SEQ ID NO: 13 [H02895_LC_VL].

In one or more embodiment(s), the heavy chain variable domain (VH) comprises the amino acid sequence of SEQ ID NO: 4 [AH02877_HC_VH] and the light chain variable domain (VL) comprises the amino acid sequence of SEQ ID NO: 14 [H02877 I106M VL].

In one or more embodiment(s), the heavy chain variable domain (VH) comprises the amino acid sequence of SEQ ID NO: 4 [AH02877_HC_VH] and the light chain variable domain (VL) comprises the amino acid sequence of SEQ ID NO: 15[AH02877_l106V_VL].

In one or more embodiment(s), the heavy chain variable domain (VH) comprises the amino acid sequence of SEQ ID NO: 4 [AH02877_HC_VH] and the light chain variable domain (VL) comprises the amino acid sequence of SEQ ID NO: 17 [AHO2877_I1O6M_V11OD_VL],

In one or more embodiment(s), the heavy chain variable domain (VH) comprises the amino acid sequence of SEQ ID NO: 4 [AH02877_HC_VH] and the light chain variable domain (VL) comprises the amino acid sequence of SEQ ID NO: 18 [AHO2877_I1O6V_V11OD_VL],

In one or more exemplified embodiment(s), the heavy chain variable domain (VH) comprises the amino acid sequence of SEQ ID NO: 5 [AH02879_HC_VH] and the light chain variable domain (VL) comprises the amino acid sequence of SEQ ID NO: 87 [AH02871 LC VL],

In one or more exemplified embodiment(s), the heavy chain variable domain (VH) comprises the amino acid sequence of SEQ ID NO: 5 [AH02879_HC_VH] and the light chain variable domain (VL) comprises the amino acid sequence of SEQ ID NO: 9 [AH02875 LC VL].

In one or more exemplified embodiment(s), the heavy chain variable domain (VH) comprises the amino acid sequence of SEQ ID NO: 5 [AH02879_HC_VH] and the light chain variable domain (VL) comprises the amino acid sequence of SEQ ID NO: 10 [H02877_LC_VL].

In one or more exemplified embodiment(s), the heavy chain variable domain (VH) comprises the amino acid sequence of SEQ ID NO: 5 [AH02879_HC_VH] and the light chain variable domain (VL) comprises the amino acid sequence of SEQ ID NO: 11 [H02879_LC_VL].

SUBSTITUTE SHEET (RULE 26) In one or more exemplified embodiment(s), the heavy chain variable domain (VH) comprises the amino acid sequence of SEQ ID NO: 5 [AH02879_HC_VH] and the light chain variable domain (VL) comprises the amino acid sequence of SEQ ID NO: 12 [H02886 LC VL],

In one or more exemplified embodiment(s), the heavy chain variable domain (VH) comprises the amino acid sequence of SEQ ID NO: 5 [AH02879_HC_VH] and the light chain variable domain (VL) comprises the amino acid sequence of SEQ ID NO: 13 [H02895_LC_VL].

In one or more embodiment(s), the heavy chain variable domain (VH) comprises the amino acid sequence of SEQ ID NO: 5 [AH02879_HC_VH] and the light chain variable domain (VL) comprises the amino acid sequence of SEQ ID NO: 14 [H02877 I106M VL].

In one or more embodiment(s), the heavy chain variable domain (VH) comprises the amino acid sequence of SEQ ID NO: 5 [AH02879_HC_VH] and the light chain variable domain (VL) comprises the amino acid sequence of SEQ ID NO: 15[AH02877_l106V_VL].

In one or more embodiment(s), the heavy chain variable domain (VH) comprises the amino acid sequence of SEQ ID NO: 5 [AH02879_HC_VH] and the light chain variable domain (VL) comprises the amino acid sequence of SEQ ID NO: 17 [AHO2877_I1O6M_V11OD_VL],

In one or more embodiment(s), the heavy chain variable domain (VH) comprises the amino acid sequence of SEQ ID NO: 5 [AH02879_HC_VH] and the light chain variable domain (VL) comprises the amino acid sequence of SEQ ID NO: 18 [AHO2877_I1O6V_V11OD_VL],

In one or more exemplified embodiment(s), the heavy chain variable domain (VH) comprises the amino acid sequence of SEQ ID NO: 6 [AH02886_HC_VH] and the light chain variable domain (VL) comprises the amino acid sequence of SEQ ID NO: 87 [AH02871 LC VL],

In one or more exemplified embodiment(s), the heavy chain variable domain (VH) comprises the amino acid sequence of SEQ ID NO: 6 [AH02886_HC_VH] and the light chain variable domain (VL) comprises the amino acid sequence of SEQ ID NO: 9 [AH02875 LC VL].

In one or more exemplified embodiment(s), the heavy chain variable domain (VH) comprises the amino acid sequence of SEQ ID NO: 6 [AH02886_HC_VH] and the light chain variable domain (VL) comprises the amino acid sequence of SEQ ID NO: 10 [H02877_LC_VL].

SUBSTITUTE SHEET (RULE 26) In one or more exemplified embodiment(s), the heavy chain variable domain (VH) comprises the amino acid sequence of SEQ ID NO: 6 [AH02886_HC_VH] and the light chain variable domain (VL) comprises the amino acid sequence of SEQ ID NO: 11 [H02879_LC_VL].

In one or more exemplified embodiment(s), the heavy chain variable domain (VH) comprises the amino acid sequence of SEQ ID NO: 6 [AH02886_HC_VH] and the light chain variable domain (VL) comprises the amino acid sequence of SEQ ID NO: 12 [H02886 LC VL].

In one or more exemplified embodiment(s), the heavy chain variable domain (VH) comprises the amino acid sequence of SEQ ID NO: 6 [AH02886_HC_VH] and the light chain variable domain (VL) comprises the amino acid sequence of SEQ ID NO: 13 [H02895_LC_VL].

In one or more embodiment(s), the heavy chain variable domain (VH) comprises the amino acid sequence of SEQ ID NO: 6 [AH02886_HC_VH] and the light chain variable domain (VL) comprises the amino acid sequence of SEQ ID NO: 14 [H02877 I106M VL].

In one or more embodiment(s), the heavy chain variable domain (VH) comprises the amino acid sequence of SEQ ID NO: 6 [AH02886_HC_VH] and the light chain variable domain (VL) comprises the amino acid sequence of SEQ ID NO: 15 [AH02877 I106V VL].

In one or more embodiment(s), the heavy chain variable domain (VH) comprises the amino acid sequence of SEQ ID NO: 6 [AH02886_HC_VH] and the light chain variable domain (VL) comprises the amino acid sequence of SEQ ID NO: 17 [AHO2877_I1O6M_V11OD_VL],

In one or more embodiment(s), the heavy chain variable domain (VH) comprises the amino acid sequence of SEQ ID NO: 6 [AH02886_HC_VH] and the light chain variable domain (VL) comprises the amino acid sequence of SEQ ID NO: 18 [AH02877J106V V110D_VL],

In one or more exemplified embodiment(s), the heavy chain variable domain (VH) comprises the amino acid sequence of SEQ ID NO: 7 [AH02895_HC_VH] and the light chain variable domain (VL) comprises the amino acid sequence of SEQ ID NO: 87 [AH02871 LC VL],

In one or more exemplified embodiment(s), the heavy chain variable domain (VH) comprises the amino acid sequence of SEQ ID NO: 7 [AH02895_HC_VH] and the light chain variable domain (VL) comprises the amino acid sequence of SEQ ID NO: 9 [AH02875 LC VL].

SUBSTITUTE SHEET (RULE 26) In one or more exemplified embodiment(s), the heavy chain variable domain (VH) comprises the amino acid sequence of SEQ ID NO: 7 [AH02895_HC_VH] and the light chain variable domain (VL) comprises the amino acid sequence of SEQ ID NO: 10 [H02877_LC_VL].

In one or more exemplified embodiment(s), the heavy chain variable domain (VH) comprises the amino acid sequence of SEQ ID NO: 7 [AH02895_HC_VH] and the light chain variable domain (VL) comprises the amino acid sequence of SEQ ID NO: 11 [H02879_LC_VL].

In one or more exemplified embodiment(s), the heavy chain variable domain (VH) comprises the amino acid sequence of SEQ ID NO: 7 [AH02895_HC_VH] and the light chain variable domain (VL) comprises the amino acid sequence of SEQ ID NO: 12 [H02886 LC VL].

In one or more exemplified embodiment(s), the heavy chain variable domain (VH) comprises the amino acid sequence of SEQ ID NO: 7 [AH02895_HC_VH] and the light chain variable domain (VL) comprises the amino acid sequence of SEQ ID NO: 13 [H02895_LC_VL].

In one or more embodiment(s), the heavy chain variable domain (VH) comprises the amino acid sequence of SEQ ID NO: 7 [AH02895_HC_VH] and the light chain variable domain (VL) comprises the amino acid sequence of SEQ ID NO: 14 [H02877 I106M VL].

In one or more embodiment(s), the heavy chain variable domain (VH) comprises the amino acid sequence of SEQ ID NO: 7 [AH02895_HC_VH] and the light chain variable domain (VL) comprises the amino acid sequence of SEQ ID NO: 15 [AH02877 I106V VL].

In one or more embodiment(s), the heavy chain variable domain (VH) comprises the amino acid sequence of SEQ ID NO: 7 [AH02895_HC_VH] and the light chain variable domain (VL) comprises the amino acid sequence of SEQ ID NO: 17 [AHO2877_I1O6M_V110D_VL],

In one or more embodiment(s), the heavy chain variable domain (VH) comprises the amino acid sequence of SEQ ID NO: 7 [AH02895_HC_VH] and the light chain variable domain (VL) comprises the amino acid sequence of SEQ ID NO: 18 [AH02877J106V V110D_VL],

V110D variants

SEQ ID NO: 16 have been predicted to have a lower overall self-adjusted immunogenicity risk score.

SUBSTITUTE SHEET (RULE 26) Thus, in a preferred embodiment, the light chain variable domain (VL) comprises the amino acid sequence of SEQ ID NO: 16 [AH02877 V110D VL], or a functional homologue thereof having an amino acid sequence which is at least 80 % identical, such as at least 81 %, 82 %, 83 %, 84 %, 85 %, 86 %, 87 %, 88 %, 89 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 95 %, 96 %, 97 %, 98 %, 99 % or 99.9 % identical to any of the variants of SEQ ID NO: 16.

In one or more preferred embodiment(s), the heavy chain variable domain (VH) comprises the amino acid sequence of SEQ ID NO: 2 [AH02871_HC_VH] and the light chain variable domain (VL) comprises the amino acid sequence of SEQ ID NO: 16 [AH02877 V110D VL].

In one or more preferred embodiment(s), the heavy chain variable domain (VH) comprises the amino acid sequence of SEQ ID NO: 3 [AH02875_HC_VH] and the light chain variable domain (VL) comprises the amino acid sequence of SEQ ID NO: 16 [AH02877 V110D VL].

In one or more exemplified embodiment(s), the heavy chain variable domain (VH) comprises the amino acid sequence of SEQ ID NO: 4 [AH02877_HC_VH] and the light chain variable domain (VL) comprises the amino acid sequence of SEQ ID NO: 16 [AH02877 V110D VL].

In one or more preferred embodiment(s), the heavy chain variable domain (VH) comprises the amino acid sequence of SEQ ID NO: 5 [AH02879_HC_VH] and the light chain variable domain (VL) comprises the amino acid sequence of SEQ ID NO: 16 [AH02877 V110D VL].

In one or more preferred embodiment(s), the heavy chain variable domain (VH) comprises the amino acid sequence of SEQ ID NO: 6 [AH02886_HC_VH] and the light chain variable domain (VL) comprises the amino acid sequence of SEQ ID NO: 16 [AH02877 V110D VL].

In one or more exemplified embodiment(s), the heavy chain variable domain (VH) comprises the amino acid sequence of SEQ ID NO: 7 [AH02895_HC_VH] and the light chain variable domain (VL) comprises the amino acid sequence of SEQ ID NO: 16 [AH02877 V110D VL].

A typical antibody

A typical antibody is composed of two immunoglobulin (Ig) heavy chains and two Ig light chain domains.

SUBSTITUTE SHEET (RULE 26) Lambda or kappa light chain constant domain

In the present disclosure humanization of the light chain domain was done by grafting the CDR regions onto human light chain acceptor sequences.

Thus, in one embodiment, the antibody, antibody fragment or antibody derivative thereof comprises a lambda or kappa light chain constant domain.

In one embodiment, the antibody, antibody fragment or antibody derivative thereof is comprises a kappa light chain constant domain having an amino acid sequence of SEQ ID NO: 46, or a functional homologue thereof having an amino acid sequence which is at least 80 % identical, such as at least 81 %, 82 %, 83 %, 84 %, 85 %, 86 %, 87 %, 88 %, 89 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 95 %, 96 %, 97 %, 98 %, 99 % or 99.9 % identical to any of the variants of SEQ ID NO: 46.

In one or more embodiment(s) the light chain constant domain is of human origin.

Heavy chain constant domain

Similarly, to the light chain constant domain, humanization of the heavy chain domain was done by grafting the CDR regions onto human heavy chain acceptor sequences.

Thus, in one or more embodiment(s) of the present disclosure the antibody, antibody fragment or antibody derivative thereof is defined by i) a constant heavy chain is selected from the group consisting of IgG 1 , lgG2, lgG3 and lgG4 chain, and ii) a constant light chain is a kappa or a lambda chain, wherein heavy chain variable regions according to the present disclosure are grafted onto the constant heavy chain, and light chain variable regions according to the present disclosure are grafted onto the constant light chain, wherein the constant heavy chain comprises one or more amino acid sequences variants(s), at one or more positions selected from the group consisting of 311 , 434, 428, 438, and 435, with reference to the IgG 1 heavy chain constant domain of SEQ ID NOs: 21 [heavy chain constant domain of lgG1], or synonymous positions in lgG2, lgG3, lgG4, IgM, IgA, IgE or IgD.

IgG subtypes

SUBSTITUTE SHEET (RULE 26) In a further embodiment, the antibody, antibody fragment or antibody derivative thereof is an antibody that comprises an IgG 1 , lgG2, lgG3 or lgG4, IgM, IgA, IgE and/or IgD heavy chain constant domain of human origin.

In another embodiment, the antibody, antibody fragment or antibody derivative thereof comprises a lambda and/or kappa light chain constant domain of human origin and/or an IgG 1 , lgG2, lgG3 or lgG4, IgM, IgA, IgE or IgD heavy chain constant domain of human origin.

In another embodiment, the antibody, antibody fragment or antibody derivative thereof comprises a lambda or kappa light chain constant domain of human origin and/or an IgG 1 , lgG2, lgG3 or lgG4, IgM, IgA, IgE or IgD heavy chain constant domain of human origin.

In another embodiment, the antibody, antibody fragment or antibody derivative thereof is an antibody that comprises a lambda or kappa light chain constant domain of human origin and an IgG 1 , lgG2, lgG3 or lgG4, IgM, IgA, IgE or IgD heavy chain constant domain of human origin.

In a further embodiment, the antibody, antibody fragment or antibody derivative thereof is an antibody that comprises a kappa light chain constant domain of human origin and an IgG 1 or lgG3 heavy chain constant domain of human origin.

REW variants

Specific alterations in the heavy chain constant domain of an IgG can provide immunoglobulins with altered half-life. The present disclosure provides antibodies, antibody fragments or antibody derivatives thereof with altered binding affinity for Fc receptors, comprising at least one mutation in the Fc region of the heavy chain constant domain of the antibody, antibody fragment or antibody derivative thereof. Such variants are also described in WO2017/158426, and are located in position 311 , 434, 428, 438, and 435 of the human IgG 1 heavy chain constant domain, according to the EU sequence numbering system (EU index) as described in Kabat et al. Sequences of

SUBSTITUTE SHEET (RULE 26) Proteins of Immunological Interest, 5^th Ed, Public Health Service, Nat inst Publ Health, 1991. In specific the lgG1 Q311 R/N434W/M428E variant is referred to as REW variant, or REW mutations in the present disclosure and relates to the triple mutation Q311R, N434W and M428E in the heavy chain constant domain of the antibody, antibody fragment or antibody derivative thereof, according to the present disclosure.

In the present disclosure, the inventors show that an inclusion of the triple mutation, referred to as Q311R/N434W/M428E or REW substitution, surprisingly leads to a superior induction of ADCP- related signalling via FcyRlla-R131 was also found (see Example 4).

Q311R/N434W/M428E means that all three mutations are present, i.e. Q311R, N434W, and M428E. The chronological order is Q311R/M428E/N434W. The triple mutations can be written in any of these ways.

The antibody, antibody fragment or antibody derivative thereof comprising said REW substitutions will therefore comprise an Fc-region wherein position 311 in the Fc-region comprises an Arginine residue, position 428 in the Fc-region comprises a Glutamic acid residue, and position 434 in the Fc-region comprises a Tryptophan residue.

Thus, in one or more embodiment(s), the antibody, antibody fragment or antibody derivative thereof comprising said REW substitutions, have an enhanced and/or improved induction of antibody dependent cellular phagocytosis (ADCP).

In that regard an enhanced and/or improved induction of antibody dependent cellular phagocytosis (ADCP), may relate an at least 1 .01-fold increase, such as at least 1.1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 2, or 5-fold increase, in induction of antibody dependent cellular phagocytosis (ADCP), compared to Obinutuzumab and/or Duohexabody-CD37.

Position M252 in human IgG 1 according to the EU sequence numbering system relates to amino acid position M135 of SEQ ID NO: 21.

Position L309 in human lgG1 according to the EU sequence numbering system relates to amino acid L192 of SEQ ID NO: 21.

Position Q311 in human lgG1 according to the EU sequence numbering system relates to amino acid Q194 of SEQ ID NO: 21.

Position M428 in human IgG 1 according to the EU sequence numbering system relates to amino acid M311 of SEQ ID NO: 21.

SUBSTITUTE SHEET (RULE 26) Position H433 in human lgG1 according to the EU sequence numbering system relates to amino acid H316 of SEQ ID NO: 21.

Position N434 in human lgG1 according to the EU sequence numbering system relates to amino acid N317 of SEQ ID NO: 21.

Position Y435 in human IgG 1 according to the EU sequence numbering system relates to amino acid Y318 of SEQ ID NO: 21.

Position Q438 in human lgG1 according to the EU sequence numbering system relates to amino acid Q321 of SEQ ID NO: 21.

Thus, in one or more embodiments, the antibody, antibody fragment or antibody derivative thereof have a heavy chain constant domain that comprises one or more amino acid sequences variants(s), at one or more positions selected from the group consisting of M135, L192, Q194, N317, M311 , H316, N317, Q321 , and Y318, with reference to heavy chain constant domain of SEQ ID NO: 21 [heavy chain constant domain of lgG1 ].

In one or more embodiments, the antibody, antibody fragment or antibody derivative thereof have a heavy chain constant domain that comprise a sequence of any one of SEQ ID NOs: 22-34.

Thus, in one or more embodiments, the antibody, antibody fragment or antibody derivative thereof have a heavy chain constant domain that comprises one or more amino acid sequence variants(s), at one or more positions selected from the group consisting of Q194, N317 and M311 , with reference to heavy chain constant domain of SEQ ID NO: 21 [heavy chain constant domain of lgG1], wherein the variants are selected form the group consisting of Q194R, N317W and M311E.

In a further embodiment said one or more amino acid sequences variants(s), is/are selected from the group consisting of lgG1-Q311R/N434W/M428E, lgG1-Q311R/N434W, lgG1-Q311R, lgG1- N434W, lgG3(b)-Q311R/N434W/M428E, lgG3(b)-Q311R/N434W/Q438E/R435H, lgG1- M252S/Q311R/N434W/M428E, lgG1-Q311R/N434P/M428E, lgG1-Q311R/N434W/M428D, lgG1- Q311R/N434W/M428E/H433K, lgG1-L309K/Q311R/N434W/M428E, lgG1-

L309R/Q311R/N434W/M428E, lgG1-L309S/Q311R/N434W7/M428E, and lgG3(b)-

Q311 R/N434 W/M428E/R435H .

Example 7 shows that an antibody, antibody fragment or antibody derivative thereof may be rescued from lysosomal degradation by neonatal Fc receptor (FcRn) in human immortalized cells (HMEC-1 ) and released back into the extracellular environment, through introduction of said sequence variants. Of interest, the antibody, antibody fragment or antibody derivative thereof comprising said sequence variants were released back into the medium in higher amounts than

SUBSTITUTE SHEET (RULE 26) those lacking said variants and were not retained inside the cells in higher amounts than those lacking said sequence variants. This may indicate that an antibody, antibody fragment or antibody derivative thereof comprising said variants might be efficiently released from FcRn back into the extracellular environment during exocytosis.

In one or more embodiment(s) the antibody, antibody fragment or antibody derivative thereof, which comprises said amino acid sequence variants results in an enhanced recycling and/or reduced intracellular retention of said antibody, antibody fragment or antibody derivative thereof.

In one or more embodiments said recycling and/or intracellular retention is mediated by the neonatal Fc receptor (FcRn).

An enhanced recycling and reduced intracellular retention may result in less antibody degradation and hence a longer serum half-life of said antibody.

Thus, in one or more embodiment(s) the antibody, antibody fragment or antibody derivative thereof, which comprises said amino acid sequence variants, have an increased serum half-life, such as an at least 10 % increase, such as at least 20 %, 30 %, 40 %, 50 % or 100 % increase in serum half-life, over antibody, antibody fragment or antibody derivative thereof that lacks said amino acid sequence variants.

The REW substitutions in some cases enhances the affinity of the antibody, antibody fragment or antibody derivative thereof towards the neonatal FC receptor (FcRn), as is exemplified in example 2, where an antibody containing the REW substitutions bound reversibly to FcRn at acidic pH (6.0) with a faster on-rate and slower off-rate compared to the non REW containing variants, as well as obinutuzumab and DuoHexabody-CD37.

In an exemplified embodiment, an antibody, antibody fragment or antibody derivative thereof, comprising the REW substitutions, has an affinity (KD) for the neonatal Fc receptor (FcRn), that is more than 10-fold, such as more than 20-fold, 30-fold, 40-fold, 50-fold or 100-fold improved compared to an antibody, antibody fragment or antibody derivative thereof, having a heavy chain domain that comprises the amino acid sequence of SEQ ID NO: 42 [NNV025 HC] and a light chain domain that comprises the amino acid sequence of SEQ ID NO: 76 [NNV025 LC].

In another exemplified embodiment, an antibody, antibody fragment or antibody derivative thereof, comprising the REW substitutions, has an affinity (KD) for the neonatal Fc receptor (FcRn), that is 30-fold to 45-fold improved compared to an antibody, antibody fragment or antibody derivative thereof, having a heavy chain domain that comprises the amino acid sequence of SEQ ID NO: 42

SUBSTITUTE SHEET (RULE 26) [NNV025 HC] and a light chain domain that comprises the amino acid sequence of SEQ ID NO: 76 [NNV025 LC],

The variant Q311R in human lgG3(b) according to the EU sequence numbering system relates to amino acid position R241 of SEQ ID NO: 33 and 34.

The variant N434W in human lgG3(b) according to the EU sequence numbering system relates to amino acid position W364 of SEQ ID NO: 33 and 34.

The variant M428E in human lgG3(b) according to the EU sequence numbering system relates to amino acid position E358of SEQ ID NO: 33.

The variant R435H in human lgG3(b) according to the EU sequence numbering system relates to amino acid position H365 of SEQ ID NO: 33 and 34.

The variant Q438E in human lgG3(b) according to the EU sequence numbering system relates to amino acid position E368 of SEQ ID NO: 34.

In a preferred embodiment, the heavy chain constant domain of said antibody has an amino acid sequence selected from the group consisting of SEQ ID NOs: 22-34.

In a further preferred embodiment, the heavy chain constant region of said antibody has an amino acid sequence of any one of SEQ ID NOs: 22-34.

In a further embodiment said one or more amino acid sequences variants(s) with relation to the human lgG1 constant domain, is/are selected from the group consisting of lgG1 - Q311R/N434W/M428E, lgG1-Q311R/N434W, lgG1-Q311R, lgG1-N434W, lgG1- M252S/Q311R/N434W/M428E, lgG1-Q311R/N434P/M428E, lgG1-Q311R/N434W/M428D, lgG1- Q311R/N434W/M428E/H433K, lgG1-L309K/Q311R/N434W/M428E, lgG1- L309R/Q311R/N434W/M428E and lgG1-L309S/Q311R/N434W7M428E.

In a preferred embodiment, the antibody, antibody fragment or antibody derivative thereof is an antibody that comprises a kappa light chain constant domain of human origin and a IgG 1 heavy chain constant domain of human origin, and optionally wherein the IgG 1 heavy chain constant domain further comprises the Q194R/N317W/M311E amino acid sequence variants, with reference to SEQ ID NO: 21.

In another preferred embodiment, the antibody, antibody fragment or antibody derivative thereof is an antibody that comprises a kappa light chain constant domain of human origin and a IgG 1 heavy chain constant domain of human origin, and optionally wherein the lgG1 heavy chain constant

SUBSTITUTE SHEET (RULE 26) domain further comprises the Q311 R/N434W/M428E amino acid sequence variants, according to SEQ ID NO: 22.

In another embodiment, said one or more amino acid sequences variants(s), with relation to the human lgG3 constant domain, is/are selected from the group consisting of lgG3(b)- Q311 R/N434W/M428E, lgG3(b)-Q311 R/N434W/Q438E/R435H and lgG3(b)-

Q311 R/N434W/M428E/R435H with reference to the EU sequence numbering system.

In another embodiment, the antibody, antibody fragment or antibody derivative thereof is an antibody that comprises a kappa light chain constant domain of human origin and a lgG3(b) heavy chain constant domain of human origin, and optionally wherein the lgG3(b) heavy chain constant domain further comprises the Q311 R/N434W/M428E/R435H amino acid sequence variants, according to SEQ ID NO: 33.

In another embodiment, the antibody, antibody fragment or antibody derivative thereof is an antibody that comprises a kappa light chain constant domain of human origin and a lgG3(b) heavy chain constant domain of human origin, and optionally wherein the lgG3(b) heavy chain constant domain further comprises the Q311 R/N434W/Q438E/R435H amino acid sequence variants, according to SEQ ID NO: 34.

In a preferred embodiment, the antibody, antibody fragment or antibody derivative thereof is an antibody that comprises a kappa light chain constant domain of human origin and a IgG 1 heavy chain constant domain of human origin, and wherein the lgG1 heavy chain constant domain further comprises the Q311 R, N434W, Q311 R/N434W, M252S/Q311 R/N434W/M428E, Q311 R/N434P/M428E, Q311 R/N434W/M428D, Q311 R/N434W/M428E/H433K,

L309K/Q311 R/N434W/M428E, L309R/Q311 R/N434W/M428 or L309S/Q31 1 R/N434W7/M428E amino acid sequence variants, with reference to SEQ ID NO: 21.

In a preferred embodiment, the antibody, antibody fragment or antibody derivative thereof is an antibody that comprises a kappa light chain constant domain of human origin and a lgG3(b) heavy

SUBSTITUTE SHEET (RULE 26) chain constant domain of human origin, and wherein the lgG3(b) heavy chain constant domain further comprises the Q311R/N434W/M428E, Q311 R/N434W/M428E/R435H or

Q311 R/N434W/Q438E/R435H amino acid sequence variants, with reference to the amino acid sequence of human lgG3(b).

Thus, in one embodiment, the antibody, antibody fragment or antibody derivative thereof have a heavy chain constant domain of the amino acid sequence according to any one of SEQ ID NOs: 22-34.

Example 9 shows that the REW Fc substitutions prolongs the plasma half-life of some antibody, antibody fragment or antibody derivative thereof, in the presence of natural competition for human FcRn. In addition, the concentration of an antibody, antibody fragment or antibody derivative thereof is several folds higher in plasma over time. Therefore, Example 9 demonstrates that an antibody, antibody fragment or antibody derivative thereof of the present disclosure have a favourable in vivo profile.

Thus, in one embodiment, said one or more amino acid sequence variant(s) extends the serum half-life of the immunoglobulin as compared to an immunoglobulin lacking said mutation.

In another embodiment the antibody, antibody fragment or antibody derivative thereof comprising said amino acid sequence variants have an increased plasma half-life compared to an antibody comprising a light chain of SEQ ID NO: 37 [NNV023] and the heavy chain of SEQ ID NO: 42 and/or the therapeutic antibody selected from the group consisting of Obinutuzumab and duohexabody-CD37.

In another embodiment the antibody, antibody fragment or antibody derivative thereof comprising said Q311R/N434W/M428E amino acid sequence variants have an increased plasma half-life compared to an antibody comprising a light chain of SEQ ID NO: 37 [NNV023] and the heavy chain of SEQ ID NO: 42 and/or the therapeutic antibody selected from the group consisting of Obinutuzumab and duohexabody-CD37.

In another embodiment the antibody, antibody fragment or antibody derivative thereof comprising a light chain of SEQ ID NO: 37 [NNV029] and the heavy chain of SEQ ID NO: 43 have an increased plasma half-life compared to an antibody comprising a light chain of SEQ ID NO: 37 [NNV023] and the heavy chain of SEQ ID NO: 42 and/or the therapeutic antibody selected from the group consisting of Obinutuzumab and duohexabody-CD37.

SUBSTITUTE SHEET (RULE 26) In another embodiment the antibody, antibody fragment or antibody derivative thereof comprising a light chain of SEQ ID NO: 37 [NNV029] and the heavy chain of SEQ ID NO: 43 have an increased plasma half-life, such as but not limited to more than 1.01-fold, 2-fold, 3-fold, 4-fold, or more than 5.5-fold increased plasma half-life, compared to an antibody comprising a light chain of SEQ ID NO: 37 [NNV023] and the heavy chain of SEQ ID NO: 42 and/or the therapeutic antibody selected from the group consisting of Obinutuzumab and duohexabody-CD37.

C-terminal residue is K or absent

Removal of C-terminal Lys of the heavy chain of therapeutic antibodies reduces the number of charge variants of the molecule during the Ab processing and storage, and a natural process of Ab maturation/degradation and may contribute to maximal complement activation. Example 6 of the present disclosure indicates that removal of the C-terminal lysine does not result in any large fluctuations in thermal stability or heat induced aggregation propensity. Furthermore Examples 2 and 3 discloses that in one or more variant(s) wherein the C-terminal lysine is absent, said variant(s) does not pH-dependent FcRn binding, nor changes the ability of the variant(s) to induce ADCC. These features are favourable since, they suggest the possibility of adding the benefit of a reduced degradation and/or number of charge species, without compromising the structure/fu notion relationship of said variant.

Thus, in one embodiment, the antibody, antibody fragment or antibody derivative thereof have a heavy chain constant domain of the amino acid sequence according to any one of SEQ ID NOs: 21-34, and wherein the C-terminal residue is K or absent.

In another embodiment, the antibody, antibody fragment or antibody derivative thereof, is an antibody, wherein the C-terminal Lysine in the heavy chain constant domain according to any one of SEQ ID NOs 21-34, is absent and/or removed.

In a preferred embodiment, the antibody, antibody fragment or antibody derivative thereof have a heavy chain constant domain of the amino acid sequence according to SEQ ID NOs: 44.

In another preferred embodiment, the antibody, antibody fragment or antibody derivative thereof have a heavy chain constant domain of the amino acid sequence according to SEQ ID NOs: 44, or a functional homologue thereof having an amino acid sequence which is at least 80 % identical, such as at least 81 %, 82 %, 83 %, 84 %, 85 %, 86 %, 87 %, 88 %, 89 %, 90 %, 91 %, 92 %, 93 %,

SUBSTITUTE SHEET (RULE 26) 94 %, 95 %, 95 %, 96 %, 97 %, 98 %, 99 % or 99.9 % identical to any of the variants of SEQ ID NO: 4.

The REW variants/mutations as disclosed herein and the absent C-terminal Lysine, may also be combined, so that an antibody, antibody fragment or antibody derivative thereof, according to the present disclosure, comprises both the REW mutations and an absent C-terminal lysine.

In relation to the present disclosure, the term “absent” or “removed”, in relation to the C-terminal Lysine, relates to an amino acid sequences of a heavy chain constant domain, wherein the C- terminal residue is not lysine.

Thus, on one or more embodiments, the antibody, antibody fragment or antibody derivative thereof, comprises a heavy chain constant domain derived from the human lgG1 heavy chain constant domain, and further comprises the Q311R/N434W/M428E amino acid sequence variants, and wherein the C-terminal lysine is absent and/or removed.

In a preferred embodiment, the antibody, antibody fragment or antibody derivative thereof, comprises a heavy chain constant domain according to SEQ ID NO: 45.

Heavy chain and light chain constant domain

In a preferred embodiment, the amino acid sequence of said antibody, antibody fragment or antibody derivative thereof is a combination of heavy chain and light chain fragments, comprising,

SUBSTITUTE SHEET (RULE 26) a light chain having an amino acid sequence which is SEQ ID NO: 37 [AH02877 V110D] and a heavy chain having an amino acid sequence which is SEQ ID NO: 45 [NNV023 heavy chain].

Antibodies harbouring the beforementioned REW mutations showed superiority in inducing ADCP response via FcyRlla-R131 as shown in example 4. Where antibodies comprising a heavy chain of SEQ ID NO: 43, harbouring the REW mutations, or a heavy chain of SEQ ID NO: 45 harbouring the REW mutations and an absent C-terminal lysine, displayed a steep dose-dependent growth through all concentrations tested.

Thus, in one or more exemplified preferred embodiment(s), the amino acid sequence of said antibody, antibody fragment or antibody derivative thereof is a combination of heavy chain and light chain fragments, comprising, a light chain having an amino acid sequence which is SEQ ID NO: 37 and a heavy chain having an amino acid sequence which is SEQ ID NO: 43 [NNV029 heavy chain, AH2871+REW mutations].

In one or more further preferred exemplified embodiment(s), the amino acid sequence of said antibody, antibody fragment or antibody derivative thereof is a combination of heavy chain and light chain fragments, comprising, a light chain having an amino acid sequence which is SEQ ID NO: 37 and a heavy chain having an amino acid sequence which is SEQ ID NO: 45 [NNV031 heavy chain, AH2871+REW mutations+delCT-Lys].

As mentioned previously in the present disclosure, Example, 2 and 3 discloses that in one or more variant(s) wherein the C-terminal lysine is absent, said variant(s) does not pH-dependent FcRn binding, nor changes the ability of the variant(s) to induce ADCC. Furthermore, Example 4 discloses that similar to the REW variants, the absence of the C-terminal lysine also displayed a superior response via FcyRlla-R131.

Thus, in one or more preferred embodiment(s), the amino acid sequence of said antibody, antibody fragment or antibody derivative thereof is a combination of heavy chain and light chain fragments, comprising, a light chain having an amino acid sequence which is SEQ ID NO: 37 and a heavy chain having an amino acid sequence which is SEQ ID NO: 44 [NNV030 heavy chain, AH2871 + delCT Lys],

Monoclonal antibody

SUBSTITUTE SHEET (RULE 26) In particular, the role of monoclonal antibodies in therapies that are based on B-cell depletion, e.g. in the treatment of B-cell malignancies, has expanded since the introduction of rituximab, an antibody that is directed against the CD20 antigen on the B-cell surface.

Thus, in one or more preferred exemplified embodiment(s), the antibody, antibody fragment or antibody derivative thereof is a monoclonal antibody.

In one or more embodiment(s), the monoclonal antibody comprises the heavy chain domain of SEQ ID NO: 43 and the light chain domain of SEQ ID NO: 37.

In one or more embodiment(s), the monoclonal antibody comprises the heavy chain domain of SEQ ID NO: 43 and the light chain domain of SEQ ID NO: 38.

In one or more embodiment(s), the monoclonal antibody comprises the heavy chain domain of SEQ ID NO: 43 and the light chain domain of SEQ ID NO: 39.

In one or more embodiment(s), the monoclonal antibody comprises the heavy chain domain of SEQ ID NO: 43 and the light chain domain of SEQ ID NO: 40.

In one or more embodiment(s), the monoclonal antibody comprises the heavy chain domain of SEQ ID NO: 43 and the light chain domain of SEQ ID NO: 41 .

In one or more embodiment(s), the monoclonal antibody comprises the heavy chain domain of SEQ ID NO: 44 and the light chain domain of SEQ ID NO: 37.

In one or more embodiment(s), the monoclonal antibody comprises the heavy chain domain of SEQ ID NO: 44 and the light chain domain of SEQ ID NO: 38.

In one or more embodiment(s), the monoclonal antibody comprises the heavy chain domain of SEQ ID NO: 44 and the light chain domain of SEQ ID NO: 39.

In one or more embodiment(s), the monoclonal antibody comprises the heavy chain domain of SEQ ID NO: 44 and the light chain domain of SEQ ID NO: 40.

In one or more embodiment(s), the monoclonal antibody comprises the heavy chain domain of SEQ ID NO: 44 and the light chain domain of SEQ ID NO: 41 .

SUBSTITUTE SHEET (RULE 26) In one or more embodiment(s), the monoclonal antibody comprises the heavy chain domain of SEQ ID NO: 45 and the light chain domain of SEQ ID NO: 37.

In one or more embodiment(s), the monoclonal antibody comprises the heavy chain domain of SEQ ID NO: 45 and the light chain domain of SEQ ID NO: 38.

In one or more embodiment(s), the monoclonal antibody comprises the heavy chain domain of SEQ ID NO: 45 and the light chain domain of SEQ ID NO: 39.

In one or more embodiment(s), the monoclonal antibody comprises the heavy chain domain of SEQ ID NO: 45 and the light chain domain of SEQ ID NO: 40.

In one or more preferred embodiment(s), the monoclonal antibody comprises the heavy chain domain of SEQ ID NO: 45 and the light chain domain of SEQ ID NO: 41 .

In one or more preferred embodiment(s), the monoclonal antibody comprises the heavy chain domain of SEQ ID NO: 43 [NNV023 + HC: REW mutations] and the light chain domain of SEQ ID NO: 37 [NNV023 full light chain AH02877 V110D], or functional homologues thereof having an amino acid sequence which is at least 80 % identical, such as at least 81 %, 82 %, 83 %, 84 %, 85 %, 86 %, 87 %, 88 %, 89 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 95 %, 96 %, 97 %, 98 %, 99 % or 99.9 % identical to any of the variants of SEQ ID NO: 43 and/or SEQ ID NO: 37.

In one or more preferred embodiment(s), the monoclonal antibody comprises the heavy chain domain of SEQ ID NO: 44 and the light chain domain of SEQ ID NO: 37 [NNV023 full light chain AH02877 V110D], or functional homologues thereof having an amino acid sequence which is at least 80 % identical, such as at least 81 %, 82 %, 83 %, 84 %, 85 %, 86 %, 87 %, 88 %, 89 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 95 %, 96 %, 97 %, 98 %, 99 % or 99.9 % identical to any of the variants of SEQ ID NO: 44 and/or SEQ ID NO: 37.

In one or more preferred embodiment(s), the monoclonal antibody comprises the heavy chain domain of SEQ ID NO: 45 and the light chain domain of SEQ ID NO: 37 [NNV023 full light chain AH02877 V110D], or functional homologues thereof having an amino acid sequence which is at least 80 % identical, such as at least 81 %, 82 %, 83 %, 84 %, 85 %, 86 %, 87 %, 88 %, 89 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 95 %, 96 %, 97 %, 98 %, 99 % or 99.9 % identical to any of the variants of SEQ ID NO: 45 and/or SEQ ID NO: 37.

SUBSTITUTE SHEET (RULE 26) In one or more preferred embodiment(s), the monoclonal antibody comprises the heavy chain domain of SEQ ID NO: 43 [NNV023 + HC: REW mutations] and the light chain domain of SEQ ID NO: 37 [NNV023 full light chain AH02877_V1 10D],

In one or more preferred embodiment(s), the monoclonal antibody comprises the heavy chain domain of SEQ ID NO: 44 and the light chain domain of SEQ ID NO: 37 [NNV023 full light chain AH02877 V110D].

In one or more preferred embodiment(s), the monoclonal antibody comprises the heavy chain domain of SEQ ID NO: 45 and the light chain domain of SEQ ID NO: 37 [NNV023 full light chain AH02877 V110D].

Traditional antibody

A traditional antibody comprises two disulphide bridge linked heavy chains and two light chains, linked to the heavy chain via disulphide bridges. The heavy chain, in general comprise a variable domain (VH) and potentially three constant domains CH1-CH3, wherein CH1 and CH2 is linked via a hinge region comprising one or more cysteines partially responsible for heavy chain dimerization. CH2 and CH3 also comprises cysteines also partially responsible for heavy chain dimerization. The light chain comprises a variable domain (VL) and a constant domain (CL). The heavy chain CH1 and light chain CL are linked via one or more cysteine residues forming one or more disulphide bridge(s).

Due to the many different domains and chain linkage options several different antibody fragment variants have emerged.

Fab, Fab’, scFV, F(ab)z, F(ab)z, F(ab)s and scFv-Fc fragment.

Modifying antibody features such as molecular size, valency, binding affinity, and pharmacokinetics allows for the development of antibody fragments with tailor-made properties for a variety of clinical applications. Variation in molecular size and binding properties among antibody fragments and antibody derivates thereof is considered to possess a central role in the tissue distribution of targeting molecules.

Thus, in one or more embodiment(s), the antibody, antibody fragment or antibody derivative thereof is a fragment selected from the group consisting of a Fab, Fab’, scFV, F(ab’)2, F(ab)2, F(ab)s and scFv-Fc fragment.

SUBSTITUTE SHEET (RULE 26) Fab

The antigen-binding fragment (Fab), according to the present disclosure, is a region on an antibody that binds to antigens. It is composed of one constant and one variable domain of each of the heavy and the light chain, thus a Fab fragment does not contain an Fc fragment. The variable domains comprises the antigen-binding site, comprising the CDRs.

Fab fragments may be prepared from an IgG like antibody by enzymatic degradation targeting the hinge region of said antibody. Alternatively, a Fab fragment is produced in a host cell, comprising a nucleotide encoding the Fab fragment, thus, only producing the portion of the antibody fragment that is the Fab fragment.

Thus, in one or more embodiments, the antibody, antibody fragment or antibody derivative thereof is a Fab fragment, comprising a heavy chain variable domain (VH) that comprises any one of the amino acid sequence variants of SEQ ID NO: 1 [heavy chain of H02871 w variants] and the light chain variable domain (VL) comprises any one of the amino acid sequence variants of SEQ ID NO: 8 [light chain of H02871 w variants], or functional homologues thereof having an amino acid sequence which is at least 80 % identical, such as at least 81 %, 82 %, 83 %, 84 %, 85 %, 86 %, 87 %, 88 %, 89 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 95 %, 96 %, 97 %, 98 %, 99 % or 99.9 % identical to any of the variants of SEQ ID NO: 1 and/or SEQ ID NO: 8.

Thus, in one or more further embodiments, the antibody, antibody fragment or antibody derivative thereof is a Fab fragment, comprising a heavy chain variable domain (VH) that comprises the amino acid sequence of SEQ ID NO: 2 [heavy chain of H02871 w variants] and the light chain variable domain (VL) comprises the amino acid sequence of SEQ ID NO: 87 [light chain of H02871 w variants].

Fab’

A Fab’ fragment, according to the present disclosure, is a Fab fragment, further comprising at least a portion of the hinge region of a traditional antibody, but which does not comprise a disulphide bridge responsible for dimerization of the individual fragments.

Fab’ fragments may be prepared from an IgG like antibody by enzymatic degradation targeting the hinge region of said antibody. Alternatively, a Fab’ fragment is produced in a host cell, comprising a nucleotide encoding the Fab’ fragment, thus, only producing the portion of the antibody fragment that is the Fab’ fragment, wherein the Fab’ fragment is postprocessed in order to reduce disulphide bridges formed between individual Fab’ fragments.

SUBSTITUTE SHEET (RULE 26) Thus in one or more embodiments, the antibody, antibody fragment or antibody derivative thereof is a Fab’ fragment, comprising a heavy chain variable domain (VH) that comprises any one of the amino acid sequence variants of SEQ ID NO: 1 [heavy chain of H02871 w variants] and the light chain variable domain (VL) comprises any one of the amino acid sequence variants of SEQ ID NO: 8 [light chain of H02871 w variants], or functional homologues thereof having an amino acid sequence which is at least 80 % identical, such as at least 81 %, 82 %, 83 %, 84 %, 85 %, 86 %, 87 %, 88 %, 89 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 95 %, 96 %, 97 %, 98 %, 99 % or 99.9 % identical to any of the variants of SEQ ID NO: 1 and/or SEQ ID NO: 8.

Thus, in one or more further embodiments, the antibody, antibody fragment or antibody derivative thereof is a Fab’ fragment, comprising a heavy chain variable domain (VH) that comprises the amino acid sequence of SEQ ID NO: 2 [heavy chain of H02871 w variants] and the light chain variable domain (VL) comprises the amino acid sequence of SEQ ID NO: 87 [light chain of H02871 w variants].

F(ab)₂,

A Fab’ fragment, according to the present disclosure, is a Fab fragment, further comprising at least a portion of the hinge region of a traditional antibody i.e., a Fab’ fragment, wherein, the disulphide bridges linking individual Fab’ fragments is not reduced, thus making a dimeric antibody Fab’ fragment, denoted as F(ab’)2.

Thus, in one or more embodiment(s), the disulphides of the antibody fragment hinge region is reduced, resulting in a Fab’ antibody fragment. In one or more embodiment(s) the disulphides are oxidized resulting in a F(ab’)2 antibody fragment.

In that regard, in one or more embodiments, the antibody, antibody fragment or antibody derivative thereof is a F(ab’)2 fragment, comprising one or more heavy chain variable domain(s) (VH) that comprises any one or more of the amino acid sequence variants of SEQ ID NO: 1 [heavy chain of H02871 w variants] and one or more light chain variable domain(s) (VL) comprises any one or more of the amino acid sequence variants of SEQ ID NO: 8 [light chain of H02871 w variants], or functional homologues thereof having an amino acid sequence which is at least 80 % identical, such as at least 81 %, 82 %, 83 %, 84 %, 85 %, 86 %, 87 %, 88 %, 89 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 95 %, 96 %, 97 %, 98 %, 99 % or 99.9 % identical to any of the variants of SEQ ID NO: 1 and/or SEQ ID NO: 8.

SUBSTITUTE SHEET (RULE 26) Thus, in one or more further embodiments, the antibody, antibody fragment or antibody derivative thereof is a F(ab’)2 fragment, comprising a heavy chain variable domain (VH) that comprises the amino acid sequence of SEQ ID NO: 2 [heavy chain of H02871 w variants] and a light chain variable domain (VL) comprises the amino acid sequence of SEQ ID NO: 87 [light chain of H02871 w variants].

Alternatively, the Fab or Fab’ fragments are chemically linked, by a chemical linker, such as but not limited to a disuccinimidyl suberate (DSS) linker, N-Hydroxysuccinimide-Polyethyleneglycol (NHS- PEG) linker or similar chemical linker, resulting in a chemically linked F(ab)2 or F(ab’)2 fragment.

In that regard the F(ab)2 or F(ab’)2 may be a monospecific or bi-specific antibody fragment.

F(ab)₃

Fab, Fab’, F(ab)2 or F(ab’)2 may also be combined into a F(ab)3 fragment comprising three individual Fab or Fab’, or a F(ab)2, a F(ab’)2 and a Fab or Fab’ fragment, thus, comprising three light chains and three heavy chain fragments, linked into a tripart fragment.

The F(ab)3 fragment may be assembled by disulphide linkage or chemical linkage as disclosed herein.

In that regard, in one or more embodiments, the antibody, antibody fragment or antibody derivative thereof is a F(ab)3 fragment, comprising one or more heavy chain variable domain (VH) that comprises any one or more of the amino acid sequence variants of SEQ ID NO: 1 [heavy chain of H02871 w variants] and one or more light chain variable domain (VL) comprises any one or more of the amino acid sequence variants of SEQ ID NO: 8 [light chain of H02871 w variants], or functional homologues thereof having an amino acid sequence which is at least 80 % identical, such as at least 81 %, 82 %, 83 %, 84 %, 85 %, 86 %, 87 %, 88 %, 89 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 95 %, 96 %, 97 %, 98 %, 99 % or 99.9 % identical to any of the variants of SEQ ID NO: 1 and/or SEQ ID NO: 8.

Thus, in one or more further embodiments, the antibody, antibody fragment or antibody derivative thereof is a F(ab)s fragment, comprising a heavy chain variable domain (VH) that comprises the amino acid sequence of SEQ ID NO: 2 [heavy chain of H02871 w variants] and a light chain variable domain (VL) comprises the amino acid sequence of SEQ ID NO: 87 [light chain of H02871 w variants].

SUBSTITUTE SHEET (RULE 26) In that regard the F(ab)s may be a monospecific, bi-specific or trispecific antibody fragment. scFV

An scFV fragment, according to the present disclosure, is an antibody fragment, comprising a heavy chain fragment comprising a variable domain (VH) and optionally a constant domain (CH) and a light chain fragment comprising a variable domain (VL) and optionally a constant light chain domain (CL), wherein the light chain and the heavy chain fragments are linked by a linker, thus making a single fragment. Such as linker may be a chemical linker as described in the present disclosure, an amino acid linker, such as but not limited to a poly-Gly-Ser linker or it may be a combination of a chemical and amino acid linker.

In that regard, in one or more embodiments, the antibody, antibody fragment or antibody derivative thereof is a scFV fragment, comprising a heavy chain variable domain (VH) that comprises any one of the amino acid sequence variants of SEQ ID NO: 1 [heavy chain of H02871 w variants] and the light chain variable domain (VL) which comprises any one of the amino acid sequence variants of SEQ ID NO: 8 [light chain of H02871 w variants], or functional homologues thereof having an amino acid sequence which is at least 80 % identical, such as at least 81 %, 82 %, 83 %, 84 %, 85 %, 86 %, 87 %, 88 %, 89 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 95 %, 96 %, 97 %, 98 %, 99 % or 99.9 % identical to any of the variants of SEQ ID NO: 1 and/or SEQ ID NO: 8, wherein the fragment further comprises a linker linking the heavy chain and the light chain.

Thus, in one or more further embodiments, the antibody, antibody fragment or antibody derivative thereof is an scFV fragment, comprising a heavy chain variable domain (VH) that comprises the amino acid sequence of SEQ ID NO: 2 [heavy chain of H02871 w variants] and a light chain variable domain (VL) comprises the amino acid sequence of SEQ ID NO: 87 [light chain of H02871],

Individual scFV fragment may also be linked to form a dimeric, trimeric or tetrameric scFV construct i.e., scFVz, scFVs, scFV4. Such a link may also be a disulphide link. scFVz, scFVs, scFV4 are also referred to as diabodies, tribadies or tetrabodies.

Diabody, tribody or tetrabody

Thus, in yet another embodiment, the antibody, antibody fragment or antibody derivative thereof the antibody fragment is a diabody, triabody, or tetrabody.

SUBSTITUTE SHEET (RULE 26) scFv-Fc

An scFV fragment, according to the present disclosure, is an antibody fragment, comprising a heavy chain fragment comprising a variable domain (VH) and more than one constant domain (CH), such as but not limited to CHi and CH2 or CH1, CH2 and CH3, and a light chain fragment comprising a variable domain (VL) and optionally a constant light chain domain (CL), wherein the light chain and the heavy chain fragments are linked by a linker, thus making a single fragment. Such as linker may be a chemical linker as described in the present disclosure, an amino acid linker, such as but not limited to a poly-Gly-Ser linker or it may be a combination of a chemical and amino acid linker.

In that regard, in one or more embodiments, the antibody, antibody fragment or antibody derivative thereof is a scFV-Fc fragment, comprising a heavy chain variable domain (VH) that comprises any one of the amino acid sequence variants of SEQ ID NO: 1 [heavy chain of H02871 w variants] and the light chain variable domain (VL) which comprises any one of the amino acid sequence variants of SEQ ID NO: 8 [light chain of H02871 w variants], or functional homologues thereof having an amino acid sequence which is at least 80 % identical, such as at least 81 %, 82 %, 83 %, 84 %, 85 %, 86 %, 87 %, 88 %, 89 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 95 %, 96 %, 97 %, 98 %, 99 % or 99.9 % identical to any of the variants of SEQ ID NO: 1 and/or SEQ ID NO: 8, wherein the fragment further comprises a linker linking the heavy chain and the light chain.

Thus, in one or more further embodiments, the antibody, antibody fragment or antibody derivative thereof is an scFV-Fc fragment, comprising a heavy chain variable domain (VH) that comprises the amino acid sequence of SEQ ID NO: 2 [heavy chain of H02871 w variants] and a light chain variable domain (VL) comprises the amino acid sequence of SEQ ID NO: 87 [light chain of H02871],

Minibody

A scFV-Fc fragment comprising a CH1 and CH2 domain may also be referred to as a minibody.

Thus, in one or more embodiment(s), the antibody, antibody fragment or antibody derivative thereof the antibody fragment is a minibody.

An anti-CD37 antibody

The present disclosure relates to an antibody, antibody fragment or antibody derivative thereof, wherein the antibody, antibody fragment or antibody derivative thereof is optimized for binding to

SUBSTITUTE SHEET (RULE 26) CD37. Binding of a CD37-specific antibody, antibody fragment or antibody derivative thereof, to cancer cells may trigger various mechanisms of action:

- After the antibody, antibody fragment or antibody derivative thereof, binds to the extracellular domain of the CD37 antigen, it may activate the complement cascade and lyse the targeted cell.

- An anti-CD37 antibody, antibody fragment or antibody derivative thereof, may mediate antibody-dependent cell-mediated cytotoxicity (ADCC), antibody dependent cellular phagocytosis (ADCP) and/or complement-dependent cytotoxicity (CDC), to the target cell, which occurs after the Fc portion of the bound antibody is recognized by appropriate receptors on cytotoxic cells of the immune system.

- The antibody may alter the ability of B-cells to respond to antigen or other stimuli.

- Finally, anti-CD37 antibody, antibody fragment or antibody derivative thereof, may initiate programmed cell death (apoptosis).

Thus, in one embodiment, the antibody, antibody fragment or antibody derivative thereof is a CD37 targeting molecule.

In one embodiment, the antibody, antibody fragment or antibody derivative thereof is an optimized CD37 targeting molecule.

In one embodiment, the antibody, antibody fragment or antibody derivative thereof comprises an optimized CD37 targeting light chain.

In one embodiment, the antibody, antibody fragment or antibody derivative thereof is an anti-CD37 antibody, antibody fragment or antibody derivative thereof according to the present disclosure.

In another embodiment, the antibody, antibody fragment or antibody derivative thereof is a polyclonal anti-CD37 antibody.

In one or more exemplified embodiment(s), the antibody, antibody fragment or antibody derivative thereof is a monoclonal anti-CD37 antibody.

In one or more embodiment(s) the antibody, antibody fragment or antibody derivative thereof, forms IgG complexes in the presence of CD37.

SUBSTITUTE SHEET (RULE 26) A derivate thereof

In the present disclosure an antibody derivate relates to antibody derivatives that make use of selected parts of an antibody resulting in molecules with novel biological activity and rationally designed mechanisms of action.

In that regard, in one or more embodiment(s) of the present disclosure relates to an antibody derivate.

Human or humanized antibody

Glycosylation

Antibody glycosylation defines the functional potential of the antibody by delineating the structure of the antibody Fc region and determining which Fc receptors it can bind to in order to recruit effector cells.

The effector functions that antibodies mediate, including cytotoxicity and phagocytosis, are critical for protection against and prevention of many diseases.

Antibody glycosylation has been harnessed to improve the efficacy of monoclonal therapeutics. Antibody glycosylation can be modulated by vaccination, indicating that rational immunogen design could seek to elicit a specific antibody glycosylation response.

Thus, in one or more exemplified embodiments, the antibody, antibody fragment or antibody derivative thereof is glycosylated.

Fucose deficient

Glycoengineered therapeutic antibodies lacking core fucose residue from the Fc N-glycans exhibit strong ADCC at lower concentrations with much higher efficacy compared to fucosylated counterparts and can evade the inhibitory effect of serum immunoglobulin G (IgG) on ADCC through its high binding to gamma receptor Illa (Fc FcyRllla).

Thus, in a preferred embodiment, said glycosylation of said antibody, antibody fragment or antibody derivative thereof is fucose deficient.

SUBSTITUTE SHEET (RULE 26) In one or more embodiment(s) said fucose deficient antibody, antibody fragment or antibody derivative thereof have an enhanced and/or improved induction of antibody-dependent cell- mediated cytotoxicity (ADCC), compared to a non-fucose deficient antibody, antibody fragment or antibody derivative thereof.

Example 12 of the present disclosure shows that the afucosylated variant NNV032 has a higher potency in induction of an ADCC response compared to fucosylated variants NNV023, NNV029 and NNV031 along with Rituximab and Duohexabody-CD37.

In embodiments, an afucosylated antibody, antibody fragment or antibody derivative thereof the present invention has a potency towards induction of ADCC below 1 nM, such as below 0.9 nM, 0.8 nM, 0.7 nM, or such as below 0.6nM.

In embodiments, an afucosylated antibody, antibody fragment or antibody derivative thereof which comprises a light chain having an amino acid sequence which is SEQ ID NO: 37 and a heavy chain having an amino acid sequence which is SEQ ID NO: 45 [NNV032 heavy chain], has a potency towards induction of ADCC below 1 nM, such as below 0.9 nM, 0.8 nM, 0.7 nM, or such as below 0.6nM.

In that regard an enhanced and/or improved induction of antibody-dependent cell-mediated cytotoxicity (ADCC), may relate an at least 1.01-fold increase, such as at least 1.1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 2, or 5-fold increase, in induction of antibody-dependent cell-mediated cytotoxicity (ADCC), compared to a non-fucose deficient antibody, antibody fragment or antibody derivative thereof.

In another exemplified embodiment said fucose deficient antibody, antibody fragment or antibody derivative thereof have an enhanced and/or improved induction of antibody-dependent cell- mediated cytotoxicity (ADCC), compared to a non-fucose deficient antibody, antibody fragment or antibody derivative thereof.

Said fucose deficiency may be obtained in several ways, such as but not limited to introduction of a GDP-4-keto-6-deoxy mannose reductase of SEQ ID NO: 81 in the production host cell. This protein is a bacterial GDP-4-keto-6-deoxy mannose reductase (RMD) that depletes the cytosolic pool of GDP-4-keto-6-deoxy mannose, which is a precursor for the synthesis of fucose. This precursor is being transformed to GDP-D-Rhamnose - an important for bacteria, but inactive sugar in mammalian cell. An alternative may also be the introduction of 4-b-N-

SUBSTITUTE SHEET (RULE 26) acetylglucosaminyltransferase (GnT-lll) and Golgi a-mannosidase II (aManll), which also inhibits fucosylation, thus producing a fucose deficient product.

Thus, in one or more embodiment(s), the production host cell is engineered such that it expresses and/or overexpresses a GDP-4-keto-6-deoxy mannose reductase of SEQ ID NO: 81 and/or a 4-b- N-acetylglucosaminyltransferase (GnT-lll) and/or a Golgi a-mannosidase II (aManll).

Improved cytotoxicity

As mentioned in the present disclosure, a selection of the humanized antibodies showed the ability to induce ADCC in both Ramos and Daudi cell lines, this is exemplified in example 3 of the present disclosure. The ability of the humanized antibodies to induce ADCC was greater than the nonhumanized antibodies of the present disclosure.

In a further embodiment said antibody, antibody fragment or antibody derivative thereof have an enhanced and/or improved induction of antibody dependent cellular phagocytosis (ADCP), compared to Obinutuzumab.

In a further embodiment, said human or humanized antibody have an enhanced and/or improved induction of antibody-dependent cell-mediated cytotoxicity (ADCC), compared to a non-humanized antibody comprising the light chain of SEQ ID NO: 35 [NNV003] and the heavy chain of SEQ ID NO: 36 and/or the therapeutic antibody selected from the group consisting of Rituximab, Obinutuzumab and duohexabody-CD37.

In a further embodiment, said human or humanized antibody have an enhanced and/or improved induction of antibody dependent cellular phagocytosis (ADCP), compared to a non-humanized antibody comprising the light chain of SEQ ID NO: 35 [NNV003] and the heavy chain of SEQ ID NO: 36 and/or the therapeutic antibody selected from the group consisting of Rituximab, Obinutuzumab and duohexabody-CD37.

SUBSTITUTE SHEET (RULE 26) In a further embodiment, said human or humanized antibody have an enhanced and/or improved induction of complement-dependent cytotoxicity (CDC), compared to a non-humanized antibody comprising the light chain of SEQ ID NO: 35 [NNV003] and the heavy chain of SEQ ID NO: 36 and/or the therapeutic antibody selected from the group consisting of Rituximab, Obinutuzumab and duohexabody-CD37.

In a further embodiment, said human or humanized antibody have an enhanced and/or improved induction of complement-dependent cytotoxicity (CDC), compared to a non-humanized antibody comprising the light chain of SEQ ID NO: 35 [NNV003] and the heavy chain of SEQ ID NO: 36.

In a further embodiment, said human or humanized antibody have an enhanced and/or improved induction of complement-dependent cytotoxicity (CDC), compared to Rituximab.

In a further embodiment, said human or humanized antibody have an enhanced and/or improved induction of complement-dependent cytotoxicity (CDC), compared Obinutuzumab.

In a further embodiment, said human or humanized antibody have an enhanced and/or improved induction of complement-dependent cytotoxicity (CDC), compared duohexabody-CD37.

Example 9 shows that the performance of the NNV antibodies bearing REW mutations (NNV029 and NNV031 ) achieve higher CDC induction than Obinutuzumab in Daudi cells.

Thus, in an embodiment, said human or humanized antibody have an enhanced and/or improved induction of complement-dependent cytotoxicity (CDC), compared to Obinutuzumab in Daudi cells.

In another embodiment, said human or humanized antibody have an enhanced and/or improved induction of complement-dependent cytotoxicity (CDC), compared to Obinutuzumab, wherein the human or humanized antibody comprises an optimized CD37 targeting light chain, such as but not limited to SEQ ID NO: 37 and an IgG heavy chain comprising one or more of said REW mutations in Daudi cells.

In another embodiment, said human or humanized antibody have an enhanced and/or improved induction of complement-dependent cytotoxicity (CDC), compared to Obinutuzumab, wherein the human or humanized antibody comprises the light chain of SEQ ID NO: 37 and the heavy chain of SEQ ID NO: 43 [NNV029] or SEQ ID NO 45 [NNV031] in Daudi cells.

SUBSTITUTE SHEET (RULE 26) In a further embodiment, said human or humanized antibody have an enhanced and/or improved induction of antibody-dependent cell-mediated cytotoxicity (ADCC), compared to a non-humanized antibody comprising the light chain of SEQ ID NO: 35 [NNV003] and the heavy chain of SEQ ID NO: 36 and/or the therapeutic antibody selected from the group consisting of Rituximab, Obinutuzumab and duohexabody-CD37, in mammalian cells.

Thus, in one or more embodiment(s), the human or humanized antibody of the present disclosure have an enhanced and/or improved induction of antibody-dependent cell-mediated cytotoxicity (ADCC), compared to a non-humanized antibody comprising the light chain of SEQ ID NO: 35 [NNV003] and the heavy chain of SEQ ID NO: 36 and/or the therapeutic antibody selected from the group consisting of Rituximab, Obinutuzumab and duohexabody-CD37, optionally in Daudi and/or Ramos cells.

In a yet further embodiment, said human or humanized antibody have an enhanced and/or improved induction of antibody dependent cellular phagocytosis (ADCP), compared to a nonhumanized antibody comprising the light chain of SEQ ID NO: 35 [NNV003] and the heavy chain of SEQ ID NO: 36 and/or the therapeutic antibody Rituximab.

In a yet further embodiment, said human or humanized antibody have an enhanced and/or improved induction of complement-dependent cytotoxicity (CDC), compared to a non-humanized antibody comprising the light chain of SEQ ID NO: 35 [NNV003] and the heavy chain of SEQ ID NO: 36 and/or the therapeutic antibody Rituximab.

In a yet further embodiment, said human or humanized antibody have an enhanced and/or improved induction of antibody-dependent cell-mediated cytotoxicity (ADCC), compared to a nonhumanized antibody comprising the light chain of SEQ ID NO: 35 [NNV003] and the heavy chain of SEQ ID NO: 36 and/or the therapeutic antibody Rituximab in mammalian cells.

In a further embodiment, said human or humanized antibody have an enhanced and/or improved induction of antibody-dependent cell-mediated cytotoxicity (ADCC), compared to a non-humanized

SUBSTITUTE SHEET (RULE 26) antibody comprising the light chain of SEQ ID NO: 35 [NNV003] and the heavy chain of SEQ ID NO: 36 and/or the therapeutic antibody Rituximab, optionally in Daudi and/or Ramos cells.

An example of such an antibody is exemplified in example 3 of the present disclosure.

Rituximab

Rituximab is a chimeric monoclonal antibody targeting CD20 i.e., an anti-CD20 monoclonal chimeric antibody.

CD 20 is primarily found on the surface of immune system B cells.

Rituximab, sold under the brand name Rituxan amongst others, is a medication used to treat certain autoimmune diseases and types of cancer.

Rituximab is used for non-Hodgkin lymphoma, chronic lymphocytic leukemia, rheumatoid arthritis, granulomatosis with polyangiitis, idiopathic thrombocytopenic purpura, pemphigus vulgaris, myasthenia gravis and Epstein-Barr virus-positive mucocutaneous ulcers.

Rituximab is given by slow injection into a vein.

Rituximab is used as a comparator for several of the humanized antibodies of the present disclosure.

Obinutuzumab

Obinutuzumab (also known as afutuzumab) is a humanized anti-CD20 monoclonal antibody.

It is used as a first-line treatment for chronic lymphocytic leukemia in combination with chemotherapy or with venetoclax, as a first-line treatment for follicular lymphoma in combination with chemotherapy, and as treatment for relapsed or refractory follicular lymphoma in combination with bendamustine chemotherapy.

Obinutuzumab is used in combination with chlorambucil as a first-line treatment for chronic lymphocytic leukemia.

Obinutuzumab is used as a comparator for several of the humanized antibodies of the present disclosure.

SUBSTITUTE SHEET (RULE 26) duohexabody-CD37

Duohexabody-CD37 (Genmab) is a biparatopic anti-CD37 antibody, targeting two distinct epitopes on CD37.

Furthermore, duohexabody-CD37 comprises a E430G hexamerization-enhancing mutation.

Duohexabody-CD37 have shown great potential as a therapeutic biparatopic antibody, with high ADCC and complement-dependent cytotoxicity (CDC) activity.

Affinity for human CD37

In general, high affinity molecules are highly preferable in the development of novel therapeutics, as a high affinity may limit off-target side effects, may enhance the on-target effects and may reduce the dosage needed in order to obtain the desired effect.

Thus, in one or more embodiment(s), the antibody, antibody fragment or antibody derivative thereof has an affinity for human CD37 expressing cells below 10 nM, such as below 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM and/or such as below 1 nM, such as below 900 pM, 800 pM, 700 pM, 600 pM, 500 pM, 400 pM or 331 pM.

In one or more preferred embodiment(s), the antibody, antibody fragment or antibody derivative thereof comprises the heavy chain variable domain (VH) comprises the amino acid sequence of SEQ ID NO: 2 [AH02871_HC_VH] and the light chain variable domain (VL) comprises the amino acid sequence of SEQ ID NO: 16 [AH02877 V110D VL] and wherein the antibody, antibody fragment or antibody derivative thereof have an affinity for human CD37 expressing cells below 2 nM such as such as below 1 nM, such as below 900 pM, 800 pM, 700 pM, or 600 pM.

A nucleic acid sequence

In one or more embodiments(s) of the present disclosure a nucleic acid sequence encodes one or more amino acid sequences according to the present disclosure.

In one embodiment, the nucleic acid sequence encodes an antibody, antibody fragment or antibody derivative thereof that is a combination of heavy chain and light chain fragments, where said antibody, antibody fragment or antibody derivative comprises,

SUBSTITUTE SHEET (RULE 26) a) a light chain having an amino acid sequence which is SEQ ID NO: 37 and a heavy chain having an amino acid sequence which is SEQ ID NO: 43 [NNV029 heavy chain, AH2871+REW mutations], b) a light chain having an amino acid sequence which is SEQ ID NO: 37 and a heavy chain having an amino acid sequence which is SEQ ID NO: 44 [NNV030 heavy chain, AH2871+REW mutations], or c) a light chain having an amino acid sequence which is SEQ ID NO: 37 and a heavy chain having an amino acid sequence which is SEQ ID NO: 45 [NNV031 heavy chain, AH2871+REW mutations+delCT-Lys].

In one embodiment, a nucleic acid sequence encoding the light chain of the antibody, antibody fragment or antibody derivative thereof according to the present disclosure, encodes an amino acid sequence according to any one of SEQ ID NOs: 37-41 [amino acid sequences of light chain variants of AH02877], or a functional homologue thereof having amino acid sequence which is at least 70 % identical, such as at least 70 %, 71 %, 72 %, 73 %, 74 %, 75 %, 76 %, 77 %, 78 %, 79 %, 80 %, 81 %, 82 %, 83 %, 84 %, 85 %, 86 %, 87 %, 88 %, 89 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 95 %, 96 %, 97 %, 98 %, 99 % or 99.9 % identical to any of the variants of SEQ ID NO: 37- 41 [amino acid sequences of light chain variants of AH02877].

In one embodiment, the nucleic acid sequence encoding the heavy chain of the antibody, antibody fragment or antibody derivative thereof according to the present disclosure is a nucleic acid sequence of any one of SEQ ID NO: 47-52 [DNA encoding the heavy chains AH2871-AH2895] or a functional homologue thereof having nucleic acid sequence which is at least 70 % identical, such as at least 70 %, 71 %, 72 %, 73 %, 74 %, 75 %, 76 %, 77 %, 78 %, 79 %, 80 %, 81 %, 82 %, 83 %, 84 %, 85 %, 86 %, 87 %, 88 %, 89 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 95 %, 96 %, 97 %, 98 %, 99 % or 99.9 % identical to any of the variants of SEQ ID NO: 47-52 [DNA encoding the heavy chains AH2871-AH2895],

In a preferred embodiment, the nucleic acid sequence encoding the heavy chain of the antibody, antibody fragment or antibody derivative thereof according to the present disclosure is a nucleic acid sequence of SEQ ID NO: 47, or a functional homologue thereof having nucleic acid sequence which is at least 70 % identical, such as at least 70 %, 71 %, 72 %, 73 %, 74 %, 75 %, 76 %, 77 %, 78 %, 79 %, 80 %, 81 %, 82 %, 83 %, 84 %, 85 %, 86 %, 87 %, 88 %, 89 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 95 %, 96 %, 97 %, 98 %, 99 % or 99.9 % identical to SEQ ID NO: 47 [DNA encoding the heavy chains AH2871-AH2895].

SUBSTITUTE SHEET (RULE 26) In one embodiment, the nucleic acid sequence encoding the heavy chain of the antibody, antibody fragment or antibody derivative thereof according to the present disclosure is a nucleic acid sequence of any one of SEQ ID NO: 53-58 [DNA encoding the light chains AH2871-AH2895], or a functional homologue thereof having nucleic acid sequence which is at least 70 % identical, such as at least 70 %, 71 %, 72 %, 73 %, 74 %, 75 %, 76 %, 77 %, 78 %, 79 %, 80 %, 81 %, 82 %, 83 %, 84 %, 85 %, 86 %, 87 %, 88 %, 89 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 95 %, 96 %, 97 %, 98 %, 99 % or 99.9 % identical to any of the variants of SEQ ID NO: 53-58 [DNA encoding the light chains AH2871-AH2895],

In one embodiment, a nucleic acid sequence encoding the heavy chain of the antibody, antibody fragment or antibody derivative thereof according to the present disclosure, encodes an amino acid sequence according to any one of SEQ ID NOs: 43 or 45 [amino acid sequences of heavy chain variants of AH02871], or a functional homologue thereof having amino acid sequence which is at least 70 % identical, such as at least 70 %, 71 %, 72 %, 73 %, 74 %, 75 %, 76 %, 77 %, 78 %, 79 %, 80 %, 81 %, 82 %, 83 %, 84 %, 85 %, 86 %, 87 %, 88 %, 89 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 95 %, 96 %, 97 %, 98 %, 99 % or 99.9 % identical to any of the variants of SEQ ID NO: 43 or 45 [amino acid sequences of heavy chain REW variants of AH02871].

In one embodiment, the nucleic acid sequence encoding the heavy chain of the antibody, antibody fragment or antibody derivative thereof according to the present disclosure is a nucleic acid sequence of any one of SEQ ID NO: 88-93 [DNA encoding the heavy chains AH2871-AH2895 wo Leader seq], or a functional homologue thereof having nucleic acid sequence which is at least 70 % identical, such as at least 70 %, 71 %, 72 %, 73 %, 74 %, 75 %, 76 %, 77 %, 78 %, 79 %, 80 %, 81 %, 82 %, 83 %, 84 %, 85 %, 86 %, 87 %, 88 %, 89 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 95 %, 96 %, 97 %, 98 %, 99 % or 99.9 % identical to any of the variants of SEQ ID NO: 88-93 [DNA encoding the heavy chains AH2871-AH2895 wo Leader seq].

In one embodiment, the nucleic acid sequence encoding the light chain of the antibody, antibody fragment or antibody derivative thereof according to the present disclosure is a nucleic acid sequence of any one of SEQ ID NO: 94-99 [DNA encoding the light chains AH2871-AH2895 wo Leader seq], or a functional homologue thereof having nucleic acid sequence which is at least 70 % identical, such as at least 70 %, 71 %, 72 %, 73 %, 74 %, 75 %, 76 %, 77 %, 78 %, 79 %, 80 %, 81 %, 82 %, 83 %, 84 %, 85 %, 86 %, 87 %, 88 %, 89 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 95 %, 96 %, 97 %, 98 %, 99 % or 99.9 % identical to any of the variants of SEQ ID NO: 94-99 [DNA encoding the light chains AH2871-AH2895 wo Leader seq].

SUBSTITUTE SHEET (RULE 26) In one embodiment, the nucleic acid sequence encodes an antibody, antibody fragment or antibody derivative thereof with a variable light chain and/or variable heavy chain of any one or more of SEQ ID NOs: 1-18.

The nucleic acid sequence of the present disclosure may also comprise additional elements than the antibody, antibody fragment or antibody derivative thereof coding region. Such elements are in example, regulatory elements.

Furthermore, the host cell according to the present disclosure may comprise regulatory elements enabling the controlled over-expression of endogenous or heterologous and/or synthetic nucleic acid sequences.

The term “regulatory element", comprises promoter sequences, signal sequence, and/or arrays of transcription factor binding sites that affect transcription and/or translation of a nucleic acid sequence operably linked to the regulatory element.

Regulatory elements are found at transcriptional and post-transcriptional levels and further enable molecular networks at those levels. For example, at the post-transcriptional level, the biochemical signals controlling mRNA stability, translation and subcellular localization are processed by regulatory elements. RNA binding proteins are another class of post-transcriptional regulatory elements and are further classified as sequence elements or structural elements. Specific sequence motifs that may serve as regulatory elements are also associated with mRNA modifications. A variety of DNA regulatory elements are involved in the regulation of gene expression and rely on the biochemical interactions involving DNA, the cellular proteins that make up chromatin, gene activators and repressors, and transcription factors.

In general, the transcriptional and translational regulatory sequences include, but are not limited to, promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, binding sites for gene regulators and enhancer sequences.

Promoters and enhancers are the primary genomic regulatory components of gene expression. Promoters are DNA regions within 1-2 kilobases (kb) of a gene’s transcription start site (TSS); they contain short regulatory elements (DNA motifs) necessary to assemble RNA polymerase transcriptional machinery. However, transcription is often minimal without the contribution of DNA regulatory elements located more distal to the TSS. Such regions, often termed enhancers, are position-independent DNA regulatory elements that interact with site-specific transcription factors to establish cell type identity and regulate gene expression. Enhancers may act independently of

SUBSTITUTE SHEET (RULE 26) their sequence context and at distances of several to many hundreds of kb from their target genes through a process known as looping. Because of these features, it is difficult to identify suitable enhancers and link them to their target genes based on DNA sequence alone.

The promoter, together with other transcriptional and translational regulatory nucleic acid sequences (also termed "control sequences") is necessary to express a given gene or group of genes (an operon).

Identification of suitable promoter sequences that promotes expression of the specific gene of interest is a tedious task, which in many cases require laborious efforts. In relation to the present disclosure regulatory elements may or may not be post-translational regulators or it may or may not be translational regulators.

Thus, in one embodiment of the present disclosure the regulatory element comprises one or more elements capable of enhancing the expression, i.e. over-expression of the one or more nucleic acid sequence(s) according to the present disclosure.

The regulatory elements and the nucleic acid sequence encoding an antibody, antibody fragment or antibody derivative thereof may be combined into a single nucleic acid construct.

Nucleic acid construct

A nucleic acid construct

The nucleic acid construct may comprise at least one regulatory element that facilitates the expression of the antibody, antibody fragment or antibody derivative thereof.

The nucleic acid construct can be a recombinant nucleic acid sequence. By the term “recombinant nucleic acid sequence”, “recombinant gene/nucleic acid/DNA encoding” or "coding nucleic acid sequence" used interchangeably is meant an artificial nucleic acid sequence (i.e. produced in vitro

SUBSTITUTE SHEET (RULE 26) using standard laboratory methods for making nucleic acid sequences) that comprises a set of consecutive, non-overlapping triplets (codons) which is transcribed into mRNA and translated into a protein when under the control of the appropriate control sequences, i.e. a promoter sequence.

The boundaries of the coding sequence are generally determined by a ribosome binding site located just upstream of the open reading frame at the 5’end of the mRNA, a transcriptional start codon (AUG, GUG or UUG), and a translational stop codon (UAA, UGA or UAG). A coding sequence can include, but is not limited to, genomic DNA, cDNA, synthetic, and recombinant nucleic acid sequences.

The term "nucleic acid" includes RNA, DNA and cDNA molecules. It is understood that, as a result of the degeneracy of the genetic code, a multitude of nucleic acid sequences encoding a given protein may be produced.

A recombinant nucleic acid sequence

The recombinant nucleic sequence may be a coding DNA sequence e.g., a gene, or non-coding DNA sequence e.g., a regulatory DNA, such as a promoter sequence.

Accordingly, in one exemplified embodiment the disclosure relates to a nucleic acid construct comprising a coding nucleic sequence, i.e. a recombinant DNA sequence encoding an antibody, antibody fragment or derivate thereof, combined with a non-coding regulatory DNA sequence, e.g. a recombinant promoter DNA sequence, or a synthetic promoter sequence, wherein the coding and promoter sequences are operably linked.

The term “operably linked” refers to a functional relationship between two or more nucleic acid (e.g., DNA) segments. Operably linked refers to the functional relationship of a transcriptional regulatory sequence to a transcribed sequence. For example, a promoter sequence is operably linked to a coding sequence if it stimulates or modulates the transcription of the coding sequence in an appropriate host cell or other expression system.

Generally, promoter sequences that are operably linked to a transcribed sequence are physically contiguous to the transcribed sequence, i.e., they are c/s-acting.

In one exemplified embodiment, the nucleic acid construct of the disclosure may be a part of the vector DNA, in another embodiment the construct it is an expression cassette/cartridge that is integrated in the genome of a host cell.

SUBSTITUTE SHEET (RULE 26) Accordingly, the term “nucleic acid construct” means an artificially constructed segment of nucleic acid, in particular a DNA segment, which is intended to be 'transplanted' into a target cell, e.g. a mammalian cell, express or to modify expression of a gene/coding DNA sequence that may be included in the construct.

Integration of the nucleic acid construct of interest comprised in the construct (expression cassette) into the genome of the host cell can be achieved by conventional methods known to the skilled person.

A host cell

In general, mammalian cells are preferred for the production of therapeutic antibodies, as they produce antibodies with mammalian glycosylation patterns and generally mammalian cells are better for the production of correctly folded antibody, antibody fragment or antibody derivative thereof.

In one embodiment, the host cell is a mammalian cell selected from the group consisting of Chinese hamster ovary (CHO) cells, CHO-K1 , CHO-DG44, mouse myeloma (NSO) cells, baby hamster kidney (BHK) cells, and human embryonic kidney lines (HEK293) cells, or an insect cell.

In a preferred embodiment the host cell is a Chinese hamster ovary (CHO) cell, such as but not limited to CHO-K1 and CHO-DG44. in another embodiment, the host cell is a mouse myeloma (NSO) cell.

In yet another embodiment baby hamster kidney (BHK) cells.

In a yet further the host cell is a human embryonic kidney lines (HEK293) cell.

In a yet further the host cell is an Insect cell.

Furthermore, as described in the present disclosure, a antibody, antibody fragment or antibody derivative thereof with a humanized glycosylation pattern may be preferred, i.e., glycosylated antibody, antibody fragment or antibody derivative thereof that is fucose deficient.

In that regard, one or more embodiments relates to a host cell, wherein the cellular fucose glycosylation pathway is modified to reduce the amount of fucose in the glycosylation of said antibody, antibody fragment or antibody derivative thereof. A modified fucose glycosylation

SUBSTITUTE SHEET (RULE 26) pathway may be obtained as disclosed herein, by inclusion of one or more enzymes that modulates the fucose pathway, so that the glycosylation is fucose deficient.

Accordingly, in one embodiment, the host cell is capable of producing an antibody, antibody fragment or antibody derivative thereof according to the present disclosure, wherein the cellular fucose glycosylation pathway of said host cell is modulated, such that the host cell produces a fucose deficient antibody, antibody fragment or antibody derivative thereof.

In one or more embodiment(s), the host cell according to the present disclosure, comprises one or more nucleic acid sequence(s) encoding an antibody, antibody fragment or antibody derivative thereof according to the present disclosure.

In one or more embodiment(s), the host cell according to the present disclosure, comprises one or more nucleic acid constructs comprising at least one nucleic acid sequence encoding an antibody, antibody fragment or antibody derivative thereof according to the present disclosure.

In that regard, one or more aspect(s) of the present disclosure relates to an antibody, antibody fragment or antibody derivative thereof according to the present disclosure, produced in a host cell according to the present disclosure.

In one or more embodiment(s) the antibody, antibody fragment or antibody derivative thereof according to the present disclosure is produced in a host cell, wherein the fucose glycosylation pathway is modulated in order to produce a fucose deficient antibody, antibody fragment or antibody derivative thereof.

Leader sequence

In the production of the antibodies of the present disclosure, the production is optimized using a leader sequence inserted at the N-terminal of the amino acid sequence of the heavy chain and/or the light chain.

In one or more embodiments of the present disclosure, the leader peptide has the amino acid sequence of SEQ ID NO: 86.

SUBSTITUTE SHEET (RULE 26) In one or more embodiments of the present disclosure, the leader peptide is encoded by a nucleic acid sequence of SEQ ID NO: 100.

In that regard, in one or more embodiment(s) the the antibody, antibody fragment or antibody derivative thereof according to the present disclosure comprises a heavy chain of any one of SEQ ID NOs: 43, 44 or 45, and an N-terminal leader sequence.

In that regard, in one or more embodiment(s) the the antibody, antibody fragment or antibody derivative thereof according to the present disclosure comprise a heavy chain of any one of SEQ ID NOs: 43, 44 or 45 and an N-terminal leader sequence which is SEQ ID NO: 86.

In one or more further embodiment(s), the antibody, antibody fragment or antibody derivative thereof, comprises a heavy chain variable domain (VH) that comprises any one of the amino acid sequence variants of SEQ ID NO: 1 [heavy chain of H02871 w variants] and the light chain variable domain (VL) comprises any of the amino acid sequence variants of SEQ ID NO: 8 [light chain of H02871 w variants], or functional homologues thereof having an amino acid sequence which is at least 80 % identical, such as at least 81 %, 82 %, 83 %, 84 %, 85 %, 86 %, 87 %, 88 %, 89 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 95 %, 96 %, 97 %, 98 %, 99 % or 99.9 % identical to any of the variants of SEQ ID NO: 1 and/or SEQ ID NO: 8, and wherein the antibody, antibody fragment or antibody derivative thereof, further comprises an N-terminal leader sequence of SEQ ID NO: 86 and a constant heavy chain that comprises one or more amino acid sequences variants(s), at one or more positions selected from the group consisting of 311 , 434, 428, 438, and 435, with reference to the lgG1 heavy chain constant domain of SEQ ID NO: 21 [heavy chain constant domain of lgG1], or synonymous positions in lgG2, lgG3, lgG4, IgM, IgA, IgE or IgD.

An immunoconjugate

Conjugates, and specifically, Immunoconjugates are antibody, antibody fragment or antibody derivative thereof conjugated (joined) to a second molecule, usually a toxin, radioisotope or label.

Such conjugates of the antibody, antibody fragment or antibody derivative thereof are all aspects of the present disclosure.

Thus, one or more aspect(s) of the present disclosure relates to an antibody, antibody fragment or antibody derivative thereof, drug conjugate that binds to human CD37 comprising: a) an antibody, antibody fragment or antibody derivative thereof according to the present disclosure, b) a linker, and

SUBSTITUTE SHEET (RULE 26) c) a drug selected from the group consisting of a toxin, a radioisotope, an anticancer drug, a cytotoxic drug and a cytostatic drug.

Chelating linkers are discussed in the below section regarding radioimmunoconjugates and the chelating linkers described therein are therefore all considered useful for conjugates comprising an antibody, antibody fragment or antibody derivative thereof of the present disclosure connected to or associated with a chelating linker.

Thus, in one or more embodiment(s), said linker is a chelating linker.

A toxin

An immunotoxin is a human-made protein that consists of a targeting portion such as an antibody, linked to a toxin. When the protein binds to that cell, it is taken in through endocytosis or similar pathway, and the toxin kills the cell.

These immunotoxins are usually used for the treatment of some kinds of cancer and a few viral infections.

These proteins are usually made of a modified antibody or antibody fragment, attached to a fragment of a toxin.

The targeting portion is composed of the Fv portion of an antibody that targets a specific cell type. In that sense the targeting portion may be an antibody, antibody fragment or antibody derivative thereof according to the present disclosure.

The toxin is usually a cytotoxic protein or compound derived from a bacterial or plant protein or of synthetic origin, from which the natural binding domain has been removed so that the Fv directs the toxin to the antigen on the target cell.

In one or more embodiment(s) the toxin is a chemotherapeutic molecule, including, but not limited to alkylating agents (cisplatin, carboplatin, oxaliplatin, mechlorethamine, cyclophosphamide, chlorambucil, ifosfamide), anti-metabolites (azathioprine, mercaptopurine, pyrimidines), alkaloids (vincristine, vinblastine, cinorelbine, vindesine, paclitaxel, docetaxel, etoposide, teniposide),

SUBSTITUTE SHEET (RULE 26) topoisomerase inhibitors (irinotecan, topotecan, amascrine, etoposide, teniposide) and cytotoxic antibiotics (actinomycin, doxorubicin, daunorubicin, calrubicin, idarubicin, epirubicin, bleomycin, plicamycin, mitomycin).

In one embodiment doxorubicin is conjugated to the antibody, antibody fragment or antibody derivative thereof via the cross-linker SMCC-hydrazide (4-[N-maleimidomethyl]cyclohexane-1- carboxyl hyd razid e) .

The immunotoxin works by the antibody (or other targeting moiety) binding to an antigen on the target cell followed by toxin that enters and kills the cell.

Thus, an aspect of the present disclosure relates to an immunotoxin that comprises antibody, antibody fragment or antibody derivative thereof according to the present disclosure.

An aspect of the present disclosure relates to an antibody, antibody fragment or antibody derivative thereof, drug conjugate that binds to human CD37 according to present invention, coupled or linked to an anticancer drug, a cytotoxic drug, or a cytostatic drug.

Drug is a radionuclide

An aspect of the present disclosure relates to a radioimmunoconjugate that binds human CD37 comprising an antibody, antibody fragment or antibody derivative thereof according to the present disclosure, a linker, and a radionuclide selected from the group consisting of ²¹¹At, ²¹³Bi, ²¹²Bi, ²¹²Pb, ²²⁵Ac, ²²⁷Th, ⁹⁰Y, ¹⁶¹Tb, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁹⁹Au, ¹⁹⁴lr, ¹⁶⁶Ho, ¹⁵⁹Gd, ¹⁵³Sm, ¹⁴⁹Pm, ¹⁴²Pr, ¹¹¹Ag, ¹⁰⁹Pd, ⁷⁷ As, ⁶⁷Cu, ⁶⁴Cu, ⁴⁷Sc, and ¹⁷⁷Lu.

In a preferred embodiment, said drug is a radionuclide, selected from the group consisting of ¹⁷⁷Lu, ²²⁵Ac, ²²⁷Th, ²¹²Pb and ⁹⁰Y.

In another embodiment of the present disclosure the radionuclide is ¹⁷⁷Lu.

In another embodiment of the present disclosure the radionuclide is ²¹²Pb.

In yet another embodiment the radionuclide is another beta-emitter or an alpha-emitter.

SUBSTITUTE SHEET (RULE 26) The radionuclide may be attached to the antibody by first reacting a bifunctional chelator, e.g., p- SCN-bn-DOTA (Macrocyclics, Tx, USA), with the antibody, followed by purification to remove unconjugated chelator, and then reaction of the chelator antibody conjugate with the radionuclide, followed by purification to remove any unconjugated radionuclide.

Alternatively, the chelator and the radionuclide can be combined firstly and subsequently conjugated to the antibody.

Chelating linkers like, e.g., p-SCN-bn-DOTA, can be used for conjugating other metal radionuclides to the antibody, antibody fragment or antibody derivative thereof according to the present disclosure.

Any type of linker with sufficient complexing ability and a functional group allowing direct or indirect conjugation to a protein or a peptide could be used.

Examples of such linkers are described in the literature (e.g. Brechbiel, 2008; Liu, 2008). Some useful examples are bifunctional cyclic chelators like p-SCN-bn-DOTA, DOTA-NHS-ester, p-SCN- Bn-TCMC; bifunctional linear chelators like p-SCN-Bn-DTPA and CHX-A"-DTPA.

The radionuclides in the present disclosure will preferably be conjugated to a targeting molecule by using bifunctional chelators.

These could be cyclic, linear or branched chelators. Particular reference may be made to the polyaminopolyacid chelators which comprise a linear, cyclic or branched polyazaalkane backbone with acidic (e.g. carboxyalkyl) groups attached at backbone nitrogens.

Examples of suitable chelators include DOTA derivatives such as p-isothiocyanatobenzyl-1 ,4,7,10- tetraazacyclododecane-1 ,4,7,10-tetraacetic acid (p-SCN-Bz-DOTA) or S-2-(4- lsothiocyanatobenzyl)-1 ,4,7,10-tetra(2-carbamoylmethyl)cyclododecane and DTPA derivatives such as p-isothiocyanatobenzyl-diethylenetriaminepentaacetic acid (p-SCN-Bz-DTPA), the first being cyclic chelators, the latter linear chelators.

Metallation of the complexing moiety may be performed before or after conjugation of the complexing moiety to the targeting moiety.

The radiolabeling procedure will in general be more convenient in terms of time used etc if the chelator is conjugated to the antibody before the radiolabeling takes place.

SUBSTITUTE SHEET (RULE 26) The principles of preparing radiolabeled conjugates using chelators attached to antibodies are described broader in e.g. Liu, 2008.

Thus, an antibody, antibody fragment or antibody derivative thereof according to the present disclosure can be used to prepare radioimmunoconjugates with differences in radiation properties and effective half-lives.

For example anti-CD37 radioimmunoconjugate consisting of a an antibody comprising a light chain according to SEQ ID NO: 37 and a heavy chain according to any one of SEQ ID NOs: 43, 44 or 45, a chelating linker and a beta or alpha emitting radionuclide including, but not limited to ¹⁷⁷Lu, ²¹¹At, ²¹³Bi, ²¹²Bi, ²¹²Pb, ²²⁵Ac, ²²⁷Th, ⁹⁰Y, ¹⁶¹Tb, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁹⁹Au, ¹⁹⁴lr, ¹⁶⁶Ho, ¹⁵⁹Gd, ¹⁵³Sm, ¹⁴⁹Pm, ¹⁴²Pr, ¹¹¹Ag, ¹⁰⁹Pd, ⁷⁷ As, ⁶⁴Cu, ⁶⁷Cu, ⁴⁷Sc can be prepared and used for preparing pharmaceutical preparations and used in therapeutic applications.

A compound enriched in one or more isotopes

An aspect of the present disclosure relates to a positron emitting immunoconjugate that binds human CD37 comprising an antibody, antibody fragment or antibody derivative thereof according to the present disclosure, a linker, and a positron emitting nuclide selected from the group consisting of ¹¹C, ¹³N, ¹⁵O, ¹⁸F, ⁶⁴Cu, and ⁸⁹Zr.

Immunoconjugates may also be used in positron emission tomography.

In that sense the immunoconjugates for positron imaging are enriched in positron emitting nuclides.

The position emitting conjugates are usually prepared just prior to the imaging due to the relatively short half-life of the positron emitting nuclides.

Position emitting conjugates, usually comprises of a targeting molecule conjugated to a compound that is enriched in a positron emitting isotope.

The positron emitting nuclide may be attached to the antibody by first reacting a bifunctional chelator, e.g., p-SCN-bn-Deferoxamine, (macrocycles, US) with the antibody, followed by purification to remove unconjugated chelator, and then reaction of the chelator antibody conjugate

SUBSTITUTE SHEET (RULE 26) with the positron emitting nuclide, followed by purification to remove any unconjugated positron emitting nuclide.

Alternatively, the chelator and the positron emitting nuclide can be combined firstly and subsequently conjugated to the antibody.

Furthermore, the compound conjugated to the targeting molecule may be a compound enriched in positron emitting nuclide may be enriched in ¹¹C, ¹³N, ¹⁵O or ¹⁸F.

Chelating linkers like, e.g., p-SCN-Bn-NOTA, can be used for conjugating other metal positron emitting nuclide to an antibody, antibody fragment or antibody derivative thereof in similar fashion to that described for ⁸⁹Zr and ⁶⁴Cu.

Any type of linker with sufficient complexing ability towards the positron emitting nuclide and a functional group allowing direct or indirect conjugation to a protein or a peptide could be used.

The positron emitting nuclides of the present disclosure will preferably be conjugated to a targeting molecule by using bifunctional chelators.

A pharmaceutical composition

Antibodies, fragments and derivates thereof are usually applied in the treatment of diseases formulated in pharmaceutical compositions.

Such compositions are optimized for parameters such as physiological tolerance and shelf-life.

Thus one or more aspect(s) of the present disclosure relates to a pharmaceutical composition comprising, as the active ingredient, one or more antibody/antibodies, antibody fragment(s) or antibody derivative(s) thereof and/or an antibody, antibody fragment or antibody derivative thereof drug conjugate according to the present disclosure, and a pharmaceutically acceptable carrier.

An embodiment of the present disclosure relates to a pharmaceutical composition as described above, further comprising one or more additional therapeutic agents.

SUBSTITUTE SHEET (RULE 26) In one embodiment of the present disclosure are said one or more additional therapeutic agents are selected from agents that target a B-cell antigen other than CD37.

Such antigen may be the B-cell antigen CD20.

In another embodiment of the present disclosure are said one or more additional therapeutic agents selected from agents that induce apoptosis.

An immunotherapeutic molecule, such as an antibody, antibody fragment or antibody derivative thereof and/or conjugate thereof as described in the present disclosure, would typically be provided as a pharmaceutical composition potentially consisting of a radionuclide, according to the description above, linked via a chelator to the antibody, antibody fragment or antibody derivative thereof dissolved in a buffer solution, which to a substantial degree maintain the chemical integrity of the immunotherapeutic and/or conjugate thereof and is physiologically acceptable for infusion into patients.

Thus, an aspect of the present disclosure relates to a pharmaceutical composition comprising a antibody, antibody fragment or antibody derivative thereof according to the present disclosure, and an pharmaceutically acceptable carrier and/or excipient.

In one or more embodiment(s) of present disclosure relates to a pharmaceutical composition comprising a drug-immunoconjugate of the present disclosure, and a pharmaceutically acceptable carrier and/or excipient.

In one or more embodiment(s) of present disclosure relates to a pharmaceutical composition comprising a radioimmunoconjugate of the present disclosure, and a pharmaceutically acceptable carrier and/or excipient.

Acceptable pharmaceutical carriers include but are not limited to non-toxic buffers, fillers, isotonic solutions, etc. More specifically, the pharmaceutical carrier can be but are not limited to normal saline (0.9 %), half-normal saline, Ringer’s lactate, 5 % Dextrose, 3.3 % Dextrose/0.3 % Saline.

The physiologically acceptable carrier can contain a radiolytic stabilizer, e.g., ascorbic acid, which protect the integrity of the pharmaceutical during storage and shipment.

SUBSTITUTE SHEET (RULE 26) One embodiment of the present disclosure comprises the pharmaceutical composition of the present disclosure and one or more additional antibodies or immunoconjugates.

Antibodies include but are not limited to Rituximab, Epratuzumab, L19, F8, F16, Galiximab, Obinutuzumab Toralizumab, Alemtuzumab, Ofatumumab, Veltuzumab, Afutuzumab, DuoHexabody 37, Tositumomab, Reditux, Ibritumomab, K7153A, 37.1 and HH1.

Radioimmunoconjugates include but are not limited to Zevalin, Bexxar and Betalutin.

In another embodiment of the present disclosure one or more additional antibodies or radioimmunoconjugates targeting CD20. Antibodies include but are not limited to Rituximab, Veltuzumab, Ofatumumab, Afutuzumab, Tositumomab, Reditux and Ibritumomab.

Radioimmunoconjugates include but are not limited to Zevalin and Bexxar.

In one embodiment, said composition further comprises an additional therapeutic agent, preferably selected in the group consisting of alkylating agents, angiogenesis inhibitors, antibodies, antimetabolites, antimitotics, antiproliferatives, antivirals, aurora kinase inhibitors, apoptosis promoters (for example, Bcl-2 family inhibitors), activators of death receptor pathway, Bcr-Abl kinase inhibitors, BiTE (Bi-Specific T cell Engager) antibodies, antibody drug conjugates, biologic response modifiers, Bruton's tyrosine kinase (BTK) inhibitors, cyclin-dependent kinase inhibitors, cell cycle inhibitors, cyclooxygenase-2 inhibitors, DVDs, leukemia viral oncogene homolog (ErbB2) receptor inhibitors, growth factor inhibitors, heat shock protein (HSP)-90 inhibitors, histone deacetylase (HDAC) inhibitors, hormonal therapies, immunologicals, inhibitors of inhibitors of apoptosis proteins (lAPs), intercalating antibiotics, kinase inhibitors, kinesin inhibitors, Jak2 inhibitors, mammalian target of rapamycin inhibitors, microRNAs, mitogen-activated extracellular signal- regulated kinase inhibitors, multivalent binding proteins, non-steroidal anti-inflammatory drugs (NSAIDs), poly ADP (adenosine diphosphate )-ribose polymerase (PARP) inhibitors, platinum chemotherapeutics, polo-like kinase (Plk) inhibitors, phosphoinositide-3 kinase (PI3K) inhibitors, proteasome inhibitors, purine analogs, pyrimidine analogues, receptor tyrosine kinase inhibitors, retinoids/deltoids plant alkaloids, small inhibitory ribonucleic acids (siRNAs), topoisomerase inhibitors, ubiquitin ligase inhibitors, hypomethylating agents, checkpoints inhibitors, peptide vaccine and the like, epitopes or neoepitopes from tumor antigens, as well as combinations of one or more of these agents.

One or more aspect(s) of the present disclosure relates to a pharmaceutical composition, comprising an antibody fragment or antibody derivative thereof, or an antibody fragment or

SUBSTITUTE SHEET (RULE 26) antibody derivative thereof, drug conjugate according to the present disclosure, further comprising one or more further molecule(s), wherein the further molecules is selected from the group consisting of one or more antibodies, small molecule(s), peptide(s) and toxin(s).

One or more embodiment(s) of the present disclosure relates to a pharmaceutical composition of the present disclosure for treating B-cell malignant cells expressing the CD37 antigen.

One or more further embodiment(s) of the present disclosure relates to a pharmaceutical composition of the present disclosure for treating inflammatory disease(s) and/or autoimmune disease.

In an embodiment of the present disclosure the pharmaceutical composition is for treatment of a B- cell malignancy selected from the group consisting of B-cell non-Hodgkins lymphoma, B-cell chronic lymphocytic leukemia, hairy cell leukemia, small lymphoblastic lymphoma and multiple myeloma.

A method for producing

One or more aspect(s) of the present disclosure relates to a method for producing an antibody, antibody fragment or antibody derivative thereof according to the present disclosure, the method comprising, a) introducing into a mammalian host cell one or more nucleic acid construct(s) of the present disclosure, b) culturing said host cell in a suitable media, c) recovering said antibody, antibody fragment or antibody derivative thereof from the culturing broth, and d) purifying the antibody, antibody fragment or antibody derivative thereof. introducing into a mammalian host cell

In one embodiment of said method, the host cell is a mammalian cell selected from the group consisting of Chinese hamster ovary (CHO) cells, CHO-K1 , CHO-DG44, mouse myeloma (NS0) cells, baby hamster kidney (BHK) cells, and human embryonic kidney lines (HEK293) cells, or an insect cell.

SUBSTITUTE SHEET (RULE 26) In one embodiment of said method, the cellular fucose glycosylation pathway of said host cell is modulated, such that the host cell produces a fucose deficient antibody, antibody fragment or antibody derivative thereof.

In the present disclosure, culturing refers to the process by which cells are grown under controlled conditions, generally outside their natural environment, thus a method used to cultivate, propagate, and grow a large number of cells.

After culturing of the host cells and expression of the gene product, the purification of the protein is required; but since the vector is introduced to a host cell, the protein of interest should be recovered and/or purified from the proteins of the host cell.

Therefore, to make the purification process easy, the cloned gene could have a tag. This tag could be histidine (His) tag or any other marker peptide or protein such as but not limited to the Albumin-binding protein. As some embodiment(s) of the present disclosure relates to antibodies, antibody fragments and antibody derivates thereof, such a tag could also be the Fc domain of said antibodies, antibody fragments and antibody derivates thereof. The Fc fragment may be used as a purification tag where an interaction partner such as but not limited to immobilized protein A is used for the purification.

A method of depleting CD37 expressing B-cells

A method of treating disease

Therapeutic use of a pharmaceutical composition or solution according to the present disclosure may be for treatment against malignant cells expressing the CD37 antigen, including but not limited to a B-cell malignancy selected from the group consisting of B-cell non-Hodgkins lymphoma, B-cell chronic lymphocytic leukemia, hairy cell leukemia, lymphoplasmacytic lymphoma and multiple myeloma.

SUBSTITUTE SHEET (RULE 26) This could be described in a study with single injections of a pharmaceutical composition or solution comprising an an antibody, antibody fragment or antibody derivative thereof according to the present disclosure or Obinutuzumab in an animal model. The therapeutic efficacy of the pharmaceutical composition comprising an an antibody, antibody fragment or antibody derivative thereof according to the present disclosure is higher than a composition comprising Obinutuzumab when compared at the same amount of antibody injected, for both dosages in an intravenous Daudi lymphoma model in SCID mice.

Thus, in one or more embodiments a pharmaceutical composition or solution comprising an an antibody, antibody fragment or antibody derivative thereof according to the present disclosure improves the survival of SCID mice in an intravenous Daudi lymphoma model when compared to Obinutuzumab.

In a further embodiment a pharmaceutical composition or solution comprising an an antibody, antibody fragment or antibody derivative thereof according to the present disclosure improves the survival of SCID mice in an intravenous Daudi lymphoma model with more than 1.01-fold, 2-fold, 3- fold or more than 4-fold, when compared to Obinutuzumab.

In a further embodiment a pharmaceutical composition or solution comprising an an antibody, antibody fragment or antibody derivative thereof comprising the light chain of SEQ ID NO: 37 and the heavy chain of SEQ ID NOs: 43, 44 or 45 [NNV029 or NNV031], provides a survival of SCID mice in an intravenous Daudi lymphoma model that is higher than 30 %, such as more than 40 %, 50 % or 59 %, or such as between 30-60 %, when evaluated 11-weeks after initiation of treatment.

Other uses could be treatment of autoimmune diseases and treatment of transplantation related effects.

One or more aspect(s) of the present disclosure relates to a method of treating cancer and/or inflammatory disease(s) and/or autoimmune disease(s) comprising administering a therapeutically effective amount of an antibody, antibody fragment or antibody derivative thereof, an antibody,

SUBSTITUTE SHEET (RULE 26) antibody fragment or antibody derivative thereof, drug conjugate, or a pharmaceutical composition according to the present disclosure.

One or more aspect(s) of the present disclosure relates to a method of treating cancer comprising administering a therapeutically effective amount of an antibody, antibody fragment or antibody derivative thereof, an antibody, antibody fragment or antibody derivative thereof, drug conjugate, or a pharmaceutical composition according to the present disclosure.

One or more aspect(s) the present disclosure relates to the use of an antibody, antibody fragment or antibody derivative thereof, an antibody, antibody fragment or antibody derivative thereof, drug conjugate, or a pharmaceutical composition according to the present disclosure, in inhibiting cancer and/or inflammatory disease(s) and/or autoimmune diseases.

One or more aspect(s) of the present disclosure relates to the use of an antibody, antibody fragment or antibody derivative thereof, an antibody, antibody fragment or antibody derivative thereof drug conjugate or a pharmaceutical composition according to the present disclosure, in ameliorating cancer and/or inflammatory disease(s) and/or autoimmune diseases.

The therapy could be based on, but are not limited to, immunotherapy, beta-particle-radiation or alpha-particle-radiation or a combination of these.

The therapy could be administered either as a monotherapy or in combination with other therapies, preferentially standard treatments. Such other therapies may be pretreatment, surgery, chemotherapy (including doxorubicin, vinblastin and gemcitabine), immunotherapy, photodynamic therapy, proteasome inhibitor (including bortezomib), histone deacetylase inhibitors (including vorinostat and suberoylanilide hydroxamic acid), vitamin D3 and vitamin D3 analogs, cell cycle checkpoint inhibitors (including UCN-01 and 2-(4-(4-Chlorophenoxy)phenyl)-1H-benzimidazole-5- carboxamide), hypoxic cell radiosensitizers (including metronidazole and misonidazole), apoptosis inducers (including withaferin A) radiosensitizers, radioimmunotherapy or a combination of two or more of these.

By administered is meant intravenous infusion or intravenous injection. More specifically, the pharmaceutical composition of the present disclosure can be administered directly in a vein by a peripheral cannula connected to a drip chamber that prevents air embolism and allows an estimate of flow rate into the patient.

SUBSTITUTE SHEET (RULE 26) In one embodiment the antibody, antibody fragment or antibody derivate thereof or conjugates thereof can be administered in a repeated fashion.

In another embodiment of the present disclosure the antibody, antibody fragment or antibody derivate thereof or conjugates thereof, according to the present disclosure could be administered in a repeated fashion but with different conjugates, such as but not limited to radionuclides, e.g., beta-radioimmunotherapy could be followed by alpha-radioimmunotherapy or chemo- immunoconjugates or vice versa.

An aspect of the present disclosure relates to the use of the antibody, antibody fragment or antibody derivate thereof or conjugates thereof, of the present disclosure for the treatment of B-cell malignancies.

An embodiment of the present disclosure relates to the use of the antibody, antibody fragment or antibody derivate thereof or conjugates thereof, of the present disclosure administered in combination with or in addition to other therapy.

In an embodiment of the present disclosure the other therapies are selected from pretreatment, chemotherapy, monoclonal antibody therapy, surgery, radiotherapy, radioimmunotherapy, and/or photodynamic therapy.

In another embodiment of the present disclosure the other therapies are bone marrow transplantation or stem cell transplantation and/or therapy.

Another embodiment of the present disclosure comprises therapeutic pre-treatment using anti- CD20 and/or anti-CD37 monoclonal antibody prior to the treatment with the antibody, antibody fragment or antibody derivate thereof or conjugates thereof, of the present disclosure.

In an embodiment of the present disclosure is the pretreatment done by administering the antibody, antibody fragment or antibody derivate thereof or conjugates thereof, according to the present disclosure followed by treatment by radioimmunoconjugates of the radioimmunoconjugates of the antibody, antibody fragment or antibody derivate thereof.

An aspect of the present disclosure relates to a method for treatment of a B-cell malignancy selected from the group consisting of B-cell non-Hodgkins lymphoma, B-cell chronic lymphocytic leukemia, hairy cell leukemia, lymphoplasmacytic lymphoma and multiple myeloma, comprising administration of an effective amount of the pharmaceutical composition of the present disclosure.

SUBSTITUTE SHEET (RULE 26) In one embodiment of the present disclosure are the uses and methods of treatment of the present disclosure performed in vitro or ex vivo.

In one embodiment, the amount of the antibody fragment or antibody derivative thereof, or the antibody fragment or antibody derivative thereof, drug conjugate according to the present disclosure is at least 0.1 mg and not more than 1 g.

In an embodiment of the present disclosure the antibody, antibody fragment or antibody derivate thereof or conjugate thereof dosing is 1-1000 mg per patient, more preferably 5-50 mg per patient.

In an embodiment of the present disclosure the radioimmunoconjugate dosing is 1-1000 mg per patient, more preferably 5-50 mg per patient, and ¹⁷⁷Lu amounting to 1 - 200 MBq/kg, more preferably 10-100 MBq/kg of bodyweight.

The pharmaceutical compositions of the present disclosure comprising antibody, antibody fragment or antibody derivate thereof or conjugate thereof of the present disclosure can be used in depleting B cells that express CD37 on their surface.

In one or more aspect(s) of the present disclosure relates to an antibody, antibody fragment or antibody derivative thereof, an antibody, antibody fragment or antibody derivative thereof drug conjugate, or a pharmaceutical composition according to the present disclosure, for use as a medicament.

In one embodiment said medicament is for use in the treatment of cancer.

In a preferred embodiment said medicament is for use in the treatment of B-cell malignancies.

In example 13 of the present disclosure, the antibody variants NNV029, NNV031 and NNV032 is shown to increases the percent survival of the animals with B-cell malignancies, compared to treatment naiive animals. This highlights the suitability of antibodies of the present disclosure for treatment of B-cell malignancies. This indication is also strengthened by the data presented in example 12, which shows that two antibodies of the present invention, NNV029, NNV031 and

SUBSTITUTE SHEET (RULE 26) NNV032 induces ADCC and ADCP in cell lines mimicking both diffuse large B-cell lymphoma, Burkitt’s Lymphoma and Mantle Cell lymphoma. NNV032 induces ADCC at a higher level than NNV029 and NNV031.

Accordingly, in embodiments, an antibody, antibody fragment or antibody derivate thereof or conjugates thereof, according to the present disclosure is used in the treatment of B-cell malignancies such as but not limited to chronic lymphocytic leukemia (CLL) and non-Hodgkin lymphomas (NHL) such as diffuse large B-cell lymphoma, Burkitt’s Lymphoma and Mantle Cell lymphoma.

In addition, it is shown in example 13, that the antibody variants NNV029, NNV031 and NNV032 extents the survival and reduces body weight loss in an animal model of non-Hodgkin lymphoma, supporting the ability of NNV029, NNV031 and NNV032 to induce CDC/ADCC in vitro as indicated in examples 12.

Accordingly, in embodiments, treatment of a subject suffering from B-cell malignancies with an effective amount of an antibody, antibody fragment or antibody derivate thereof or conjugates thereof, according to the present disclosure enhances the chances of survival for said subject, compared to treatment naiive subjects suffering from B-cell malignancies.

In embodiments, an antibody, antibody fragment or antibody derivative thereof which comprises a light chain having an amino acid sequence which is SEQ ID NO: 37 and a heavy chain having an amino acid sequence which is SEQ ID NO: 45 [NNV032 heavy chain], enhances the chances of survival for said subject, compared to treatment naiive subjects suffering from B-cell malignancies.

In embodiments, an afucosylated antibody, antibody fragment or antibody derivative thereof which comprises a light chain having an amino acid sequence which is SEQ ID NO: 37 and a heavy chain having an amino acid sequence which is SEQ ID NO: 45 [NNV032 heavy chain], enhances the chances of survival for said subject, compared to treatment naiive subjects suffering from B-cell malignancies.

In additional embodiments, treatment of a subject suffering from B-cell malignancies with an effective amount of an antibody, antibody fragment or antibody derivate thereof or conjugates thereof, according to the present disclosure extends the lifetime prognosis of said subject, compared to an untreated subject suffering from B-cell malignancies.

SUBSTITUTE SHEET (RULE 26) In embodiments, an antibody, antibody fragment or antibody derivative thereof which comprises a light chain having an amino acid sequence which is SEQ ID NO: 37 and a heavy chain having an amino acid sequence which is SEQ ID NO: 45 [NNV032 heavy chain], extends the lifetime prognosis of said subject, compared to an untreated subject suffering from B-cell malignancies.

In embodiments, an afusocylated antibody, antibody fragment or antibody derivative thereof which comprises a light chain having an amino acid sequence which is SEQ ID NO: 37 and a heavy chain having an amino acid sequence which is SEQ ID NO: 45 [NNV032 heavy chain], extends the lifetime prognosis of said subject, compared to an untreated subject suffering from B-cell malignancies.

A general problem in treatment of cancers is the loss of body mass in subjects being treated. The reasons that patients are losing weight during treatment are many, and only in some cases related to the disease itself, while the method of treatment also plays a critical role in cancer treatment associated weight loss. In the present disclosure it shown, in example 13, that the antibody variants NNV029, NNV031 and NNV032 all has a reduced tendency towards weight reduction in animals undergoing treatment compared to treatment naiive animals.

Accordingly, in embodiments, treatment of a subject suffering from B-cell malignancies with an effective amount of an antibody, antibody fragment or antibody derivate thereof or conjugates thereof, according to the present disclosure reduces the weight loss of said subject, compared to a treatment naiive subject suffering from B-cell malignancies.

In embodiments, an antibody, antibody fragment or antibody derivative thereof which comprises a light chain having an amino acid sequence which is SEQ ID NO: 37 and a heavy chain having an amino acid sequence which is SEQ ID NO: 45 [NNV032 heavy chain], reduces the weight loss of said subject, compared to a treatment naiive subject suffering from B-cell malignancies.

In embodiments, an antibody, antibody fragment or antibody derivative thereof which comprises a light chain having an amino acid sequence which is SEQ ID NO: 37 and a heavy chain having an amino acid sequence which is SEQ ID NO: 43 [NNV029 heavy chain], reduces the weight loss of said subject, compared to a treatment naiive subject suffering from B-cell malignancies.

In embodiments, an afusocylated antibody, antibody fragment or antibody derivative thereof which comprises a light chain having an amino acid sequence which is SEQ ID NO: 37 and a heavy chain having an amino acid sequence which is SEQ ID NO: 45 [NNV032 heavy chain], reduces the

SUBSTITUTE SHEET (RULE 26) weight loss of said subject, compared to a treatment naiive subject suffering from B-cell malignancies.

In a preferred embodiment, said medicament is for treating of a B-cell malignancy selected from the group consisting of B-cell non-Hodgkin’s lymphoma, B-cell chronic lymphocytic leukemia, hairy cell leukemia, lymphoplasmacytic lymphoma and multiple myeloma, comprising administering to the individual in need thereof, an effective amount of an antibody, antibody fragment or antibody derivative thereof, an antibody, antibody fragment or antibody derivative thereof, drug conjugate, or a pharmaceutical composition according to the present disclosure.

In one embodiment said medicament is administered once or sequential.

One or more aspect(s) of the present disclosure relates to a formulation of an antibody, antibody fragment or antibody derivative thereof, an antibody fragment or antibody derivative thereof drug conjugate, or a pharmaceutical composition according to the present disclosure, for use in pretreatment, wherein human CD37 is blocked in normal tissues before treatment with an immunotoxic anti-CD37 molecule or antibody-drug conjugate according to the present disclosure.

Use in positron emission tomography imaging

One or more aspect(s) of the present disclosure relates to an antibody, antibody fragment or antibody derivative thereof, conjugate that binds to human CD37 comprising: a) an antibody, antibody fragment or antibody derivative thereof according to the present disclosure, b) a linker, and c) an compound enriched in one or more isotopes selected from the group consisting of 1¹C, ¹³N, ¹⁵O, ¹⁸F, and ⁸⁹Zr.

One or more aspect(s) of the present disclosure relates to an antibody, antibody fragment or antibody derivative thereof conjugate according to the present disclosure, for use in positron emission tomography imaging.

SUBSTITUTE SHEET (RULE 26) In one embodiment, said imaging is for providing diagnosis, staging, and monitoring treatment of cancers.

In another embodiment said cancer is B-cell non-Hodgkin’s lymphoma, B-cell chronic lymphocytic leukemia, hairy cell leukemia, lymphoplasmacytic lymphoma and multiple myeloma.

Kits

A kit for the production of a drug conjugate

One or more aspect(s) of the present disclosure relates to a kit for the production of an antibody fragment or antibody derivative thereof, drug conjugate according to the present disclosure comprising, a) two or more vials, wherein one vial contains a conjugate comprising a drug linked to a linker, and one vial comprising an antibody, antibody fragment or antibody derivative thereof according to the present disclosure, and b) optionally instructions for preparing said antibody-drug conjugate.

A kit for the production of a radionuclide conjugate

In the present disclosure “radioimmunoconjugate” and “radionuclide conjugate” are used interchangeably.

One or more aspect(s) of the present disclosure relates a kit for the production of an antibody fragment or antibody derivative thereof, radionuclide or positron emitting nuclide conjugate, according to the present disclosure comprising, a) two or more vials, wherein one vial contains a conjugate comprising a chelator linked to an antibody fragment or antibody derivative thereof according to the present disclosure, a second vial containing a radionuclide or positron emitting nuclide, and b) optionally, instructions for preparing said radioimmunoconjugate, or positron emitting nuclide-immunoconjugate.

One or more aspect(s) of the present disclosure relates a kit for the production of an antibody fragment or antibody derivative thereof, radioimmunoconjugate according to the present disclosure comprising,

SUBSTITUTE SHEET (RULE 26) a) two or more vials, wherein one vial contains a conjugate comprising a chelator linked to an antibody fragment or antibody derivative thereof according to the present disclosure, a second vial containing a radionuclide, and b) optionally, instructions for preparing said radioimmunoconjugate conjugate.

A kit, according to the present disclosure may require some procedures to be performed, e.g., radiolabeling and/or purification to take place before infusion.

An embodiment of the present disclosure relates to a kit of the present disclosure, wherein the content of one or several of the vials are either lyophilized or in a solution.

By mixing the contents of the two vials to generate the drug-immunoconjugate or radioimmunoconjugate the final product will appear. Thus, in another embodiment of the present disclosure the radioimmunoconjugate is generated by mixing the content of the two vials.

This product may need purification prior to use.

It should be noted that embodiments and features described in the context of one of the aspects of the present disclosure also apply to the other aspects of the disclosure.

All patent and non-patent references cited in the present application, are hereby incorporated by reference in their entirety.

The disclosure will now be described in further details in the following non-limiting examples.

SUBSTITUTE SHEET (RULE 26) EXAMPLES

In the present examples, references are made to different antibodies, which are listed in Table 1.

Table 1, antibody identifiers.

"recombined construct of two different antibody fragments, thus comprises two heavy chain sequences and two light chain sequences.

EXAMPLE 1 - Manufacturing of humanized anti-CD37 antibodies

The aim of this example is to manufacture the NNV023, NNV029, NNV030, NNV031 , NNV032, Duobody 1 and Duobody 6 antibodies. Duobody 1 and duobody 6 antibodies are precursors for assembling the duohexabody-CD37 antibody that is used as comparator for the NNV antibodies.

The present example relates to the antibodies NNV023, NNV029, NNV030, NNV031 , NNV032, Duobody 1 , doubody 6 and duohexabody-CD37.

METHODS

There were 7 samples of humanized Abs manufactured and one Ab assembled (Table 2).

Table 2. Overview of the manufactured antibodies.

During an initial experiment the following activities were performed:

1) Gene design for the variable domain and synthesis.

2) Cloning into vector system.

3) Generation of low-endotoxin plasmid DNA.

4) Pilot 10 mL transfection. 5) Analytics: DNA sequencing and titer quantification with ForteBio Protein A biosensor.

The initial experiment was followed by a larger scale expression and purification, which included these steps:

1) Preparation of transfection-quality plasmid DNA.

2) Transfection of CHO cells (supernatant generation).

3) Purification with MabSelect SuRe Protein A. NNV029, NNV031 , and NNV032 were purified using Protein G column.

4) Dilution into PBS with 100 mmol/l L-arginine (pH 6.5-6.7).

5) Sterile filtration.

6) Analytics: quantity (mg), volume (ml), concentration (mg/ml; A280 nm reading incl. extinction coefficient), purity (%; HPLC-SEC), endotoxin level (EU/mg)

Preparation of cDNA vector for transfection

The cDNAs were cloned into the vector system using conventional (non-PCR based) cloning techniques and the vector plasmids were synthesized. Plasmid DNA was prepared under low- endotoxin conditions based on anion exchange chromatography. DNA concentration was determined by measuring the absorption at a wavelength of 260 nm. Correctness of the sequences was verified with Sanger sequencing (with up to two sequencing reactions per plasmid depending on the size of the cDNA).

Cell transfection

Suspension-adapted CHO K1 cells were used for production. The seed was grown in eviGrow medium, a chemically defined, animal-component free, serum-free medium. Cells were transfected with eviFect, transfection reagent, and cells were grown after transfection in eviMake2, an animal- component-free, serum-free medium.

Purification of I gG 1s from supernatant

Supernatant was harvested by centrifugation and subsequent filtration (0.2 pm filter). The antibody was purified using regenerated (by making a low pH wash as well as a 0.1 M NaOH wash, for 1 hour in total) FPLC column filled with MabSelect™ SuRe™ alkali-tolerant ProteinA-derived affinity resin. A column with Protein G-derived affinity resin can be employed if needed.

Assembly of duohexabody-CD37

The assembly of duohexabody-CD37 was performed using a so-called “controlled Fab-arm exchange” (cFAE) approach. The cFAE allows for recombination assembly of heterodimeric bispecific antibodies. This is possible only for IgGs manufactured from parental lgG1 designed following the DuoBody technology developed at GenMab (“knob-to-hole” approach). The paranetal lgG1 antibodies were the Duobody 1 and Duobody 6 antibodies described above.

The procedure consisted of two steps: a) mixing of parental lgG1s under permissive redox conditions to yield a mix of monomeric HC+LC of lgG1 ; b) removal of the reductant to enable re-oxidation and thus assembly of the complimentary parts of duohexabody-CD37.

The precursor antibodies were mixed with 75 mM of 2-MEA, 300 min incubation at 31 °C, without shaking. Then, the buffer of the reaction mixture was exchanged to a storage buffer (0.1 M phosphate buffer, pH 7.0) using Amicon Ultra-15 centrifugal units (30-kDa MWCO). The solution was left overnight in 1.5 mL Eppendorf tube in a refrigerator for re-oxidation. Analytical methods

The concentration of Ab was determined by measuring absorption at 280 nm (Evitria). The extinction coefficient for each Ab was theoretically determined. Purity was determined by analytical size exclusion chromatography on Agilent AdvanceBio SEC column (300A 2.7 urn 7.8 x 300 mm) and DPBS as running buffer at 0.8 ml/min with detection at 280nm. Endotoxin content was measured with the Charles River Endosafe PTS system.Ab titer was measured with ForteBio Protein A biosensors (using kinetic assay method) and calculated using interpolation to a human IgG 1 standard curve. SDS-PAGE for analyzing the molecular integrity of Abs. Iso-electric focusing (IEF) in non-reducing conditions was used to determine isoelectric point (pl) and ICEX-HPLC was used to determine charge isoforms of the Abs.

RESULTS

Table 3 shows the analytical summary of the manufactured antibodies.

Table 3. Analytical summary of manufactured antibodies.

For the duohexabody-CD37 assembly six different conditions were tested with the aim to check whether different concentrations of the antibody solutions and different ratio between duobody-1 and 6 affect the assembly and quality of the antibody. Duobody-1 was not consumed when a concentration of 2mg/mL was used, but this was improved by increasing the concentration to 6 mg/ml at a 1 :1 ratio of duobody 1 and 6. There was no improvement by increasing the amount of duobody-6.

DISCUSSION Seven humanized lgG1 antibodies were manufactured. Manufacturing of NNV032 required cotransfection of CHO cell line with an additional protein, bacterial GDP-4-keto-6-deoxy mannose reductase. This protein depletes the cytosolic pool of GDP-4-keto-6-deoxy mannose, which is a precursor for the synthesis of fucose. This precursor is being transformed to GDP-D-Rhamnose - an important for bacteria, but inactive sugar in mammalian cell. Despite using the same vector and the leader sequence as for other Abs manufactured in this project, the Duobody 1 showed a lower recovery than the Duobody 6. A re-transfection for Duobody 1 with a larger volume was needed to recover the required amount of Ab. The SEC- HPLC of Duobody 1 showed around 6% of aggregates after purification (monomericity 94,5%); the Duobody 6 was just a bit better in this context, 96,8% of monomers. All other antibodies manufactured for this project were of good quality (monomers%, endotoxin level, concentration). EXAMPLE 2 - SDS-PAGE and FcRn, FcyR and C1q binding of humanized anti-CD37 antibody variants and determination of FcRn binding kinetics for humanized anti-CD37 lgG1 antibodies Therapeutic monoclonal IgG antibodies that target cancer cells benefit from their ability to interact with cell surface classical Fey receptors (FcyRs) and soluble C1q, the first component of the classical complement pathway. The interaction between antigen bound IgG and these receptors trigger downstream effector functions such as antibody dependent cellular cytotoxicity (ADCC), antibody dependent cellular phagocytosis (ADCP) and complement dependent cytotoxicity (CDC) to eliminate the target cell. The Fey receptors in humans activate effector functions upon binding to IgG. These are FcyRI and FcyRlla (H131 and R131 allotypic variants) that mediate ADCP, FcyRllla (V158 and F158 allotypic variants) that mediate ADCC and FcyRI lib that function as a decoy receptor on neutrophils. In addition, there is one inhibitory FcyR, FcyRllb. Both the FcyRs and C1q bind IgG in the lower hinge and upper CH2 domain of the constant Fc part.

In addition, IgG interacts with the neonatal Fc receptor (FcRn), a key homeostatic regulator of systemic IgG levels. FcRn binds in the CH2-CH3 elbow region of the IgG Fc in a strictly pH- dependent manner, with binding at acidic (pH 6.0) and no binding or release at neutral pH (7.4). In both hematopoietic and non-hematopoietic cells, FcRn is predominantly located within acidified endosomes, where the low pH allows binding of IgG taken up by fluid phase pinocytosis. FcRn then recycles its IgG ligand back to the cell surface for release into circulation upon exposure to the neutral pH of the blood. Antibodies that do not bind the receptor are degraded in lysosomes. The aim of the experiments is first to measure the concentration and confirm the structural integrity of a panel of engineered humanized anti-CD37 IgG 1 antibody variants (NNV) side-by-side with commercially obtained obinutuzumab (anti-CD20; IgG 1 , Gazyvaro) and a recombinant form of DuoHexabody-CD37 (anti-CD37; Biparatopic lgG1 ; DuaHexabody-CD37) by spectrophotometry as well as non-reducing and reducing SDS-PAGE. Then, the pH dependent FcRn binding properties of the antibodies are measured in ELISA and the binding kinetics at acidic pH are determined by surface plasmon resonance (SPR). The FcyR and C1q binding properties of the antibodies are also measured in ELISA.

METHODS

Measurement of antibody concentration

The concentration of the received lgG1 variants was measured on a Denovix spectrophotometer (Denovix) using the built in IgG function with an extinction coefficient of 210.000 cm^-1M^_1 (Table 4). Table 4. Measured protein concentrations.

Preparation of 1 mg/mL working stocks The received antibodies were diluted to approx. 1 mg/mL working stocks of 100 mL using 1x PBS and re-measured as above using the Denovix spectrophotometer (Table 5).

Table 5. Dilution and measurement of working stock protein concentrations.

SDS-PAGE

SDS-PAGE was performed using the Bolt MiniTank electrophoresis system (ThermoFisher) with precast 12% Bolt Bis-Tris protein gels (ThermoFisher). For non-reducing SDS-PAGE, 2 mg of each lgG1 variant was diluted in a total volume of 10 ml dPhO before addition of 2 ml 4x Bolt LDS Sample Buffer (ThermoFisher). For reducing SDS-PAGE, 2 mg of each IgG 1 variant was diluted in a final volume of 10 ml dHzO (Table 6) before addition of 2 ml 4x Bolt LDS Sample buffer and 4 ml 10x Bolt Sample Reducing Agent (ThermoFisher). Reduced samples were boiled at 95°C for 5 min. The gel was run at 200V for 22 min in 1x Bolt MES SDS running buffer (ThermoFisher) before staining with Coomassie Blue (BioRad) and repeated washing with dHzO.

Table 6. Dilution of antibody samples for SDS-PAGE

Quantification of coated lgG1 variants

96 well EIA/RIA plates (Corning Costar) were coated with 100 ml titrated amounts of IgG 1 variants (1000 - 0.45 ng/mL) diluted in phosphate buffered saline (PBS) and incubated overnight (O/N) at 4°C. Remaining surface area w blocked with PBS containing 0.05% Tween20 (T) and 4% skimmed milk powder (S) for 1 hour (h) at room temperature (RT). The plates were washed four times with PBS/T using a Hydrospeeda plate washer (Tecan). Two different detection antibodies, alkaline phosphatase (AP) conjugated anti-human IgG (Fc-specific) from goat (Sigma Aldrich) and AP conjugated anti-human kappa light chains (Sigma Aldrich) (both diluted 1 :5000 in PBS/T/S) were added to separate plates and incubated for 1 h at RT. Following washing as above, bound detection antibodies were visualized by addition of phosphatase substrate (1 mg/ml in diethanolamine buffer). The plates were developed for 20-30 min before the 405 nm absorption values were recorded using a TECAN Sunrise spectrophotometer (Tecan).

Human FcRn ELISA

96 well EIA/RIA plates were coated with 100 ml titrated amounts of IgG 1 variants (1000 - 0.45 ng/ml) diluted in PBS and incubated O/N at 4°C. Remaining surface area was blocked with PBS containing 0.05% T and 4% S for 1 h at RT. The plates were washed four times with PBS/T using a Hydrospeeda plate washer (Tecan). Preformed complexes of 250 ng/ml biotinylated soluble human FcRn and AP conjugated streptavidin (1 :1 molar ratio) were then added and incubated for 1 h at RT. Following washing as above with PBS/T or FcRn binding buffer (67 nM phosphate, 0.1 M NaCI, 0.05% T, pH 6.0), bound receptor was visualized by addition of phosphatase substrate (1 mg/ml in diethanolamine buffer). The plates were developed for 20-30 min before the 405 nm absorption values were recorded using a TECAN Sunrise spectrophotometer (Tecan).

Human FcgR ELISA 's

96 well EIA/RIA plates (CorningCostar) were coated with 100 ml titrated amounts of lgG1 variants (10.000 - 4.5 ng/ml) diluted in PBS and incubated O/N at 4°C. Remaining surface area was blocked with PBS containing 0.05% T and 4% S for 1 h at RT. The plates were washed four times with PBS/T using a Hydrospeeda plate washer (Tecan). Then 250 ng/ml biotinylated truncated soluble human FcgRI, FcgRlla-H131 , FcgRlla-R131 , FcgRIlb, FcgRllla-V158, FcgRllla-F158 or FcgRIIIb (Sino Biological), in preformed complex with AP conjugated streptavidin (GE Healthcare) (1 :1 molar ratio), were added and incubated for 1 h at RT. Following washing as above, bound receptor was visualized by addition of phosphatase substrate (1 mg/ml in diethanolamine buffer) (Sigma Aldrich). The plates were developed for 15-20 min before the 405 nm absorption values were recorded using a TECAN Sunrise spectrophotometer (Tecan). C1q ELISA

96 well EIA/RIA plates (CorningCostar) were coated with 100 ml titrated amounts of lgG1 variants (20.000 - 156.25 ng/ml) diluted in PBS and incubated O/N at 4°C. Remaining surface area was blocked with PBS containing 0.05% T and 4% S for 1 h at RT. The plates were then washed four times with PBS/T using a Hydrospeeda plate washer (Tecan). Human C1q (Complement Technologies) diluted to 360 ng/ml in veronal buffer (Complement Technologies) was added and incubated for 30 min at 37°C. Detection of bound C1q was performed using a primary anti-human C1q antibody from rabbit (DAKO) diluted 1 :10.000 in PBS/T/S and a secondary horseradish peroxidase (HRP) conjugated anti-rabbit IgG antibody diluted 1 :5000 in PBS/T/S (GE Healthcare). Binding was visualized by addition of 100 ml TMB substrate (Calbiochem) for 15-20 min before the enzymatic reaction was stopped by addition of 50 ml 1M HCI. The 450 nm absorption values were recorded using a Sunrise TECAN spectrophotometer (Tecan).

Calculation of EC50 values

EC50 values for binding of test antibodies to FcRn and FcyRs were calculated by fitting the binding data to the (agonist) v.s response (three parameters) nonlinear regression model using Graphpad Prism.

Surface plasmon resonance (SPR)

A Biacore T200 instrument (GE Healthcare) was used to couple CM5 sensor chips with IgG 1 antibody variants (300 resonance units (RU)) using amine coupling reagents provided in Immobilization Kit (GE Healthcare). Serial dilutions of monomeric His6x-tagged human FcRn (FcRn-Hisex) (1000.0 - 7.81 nM or 125.0 - 0.97 nM) were injected over the immobilized antibodies at pH 6.0 at a flow rate of 50 pl/min at 25°C. Phosphate buffer pH 6.0 (67 nM phosphate, 0.1 M NaCI, 0.005% Tween 20) was used as dilution and running buffer while HBS-P+ pH 7.4 (0,01 M HEPES, 0,15 M NaCI, 0,005% surfactant P20) (GE Healthcare) was used as regeneration buffer. For measurements of relative binding responses at pH 7.4, FcRn-His (1000 nM) was injected over the immobilized lgG1 antibody variants at a flow rate of 20 pl/min using HBS-P+ as dilution and running buffer. A zero sample and the blank reference flow cell values was subtracted from the binding data before the kinetics constants were fitted to a 1 :1 Langmuir binding model using BIAevaluation software (GE Healthcare).

RESULTS

Measurements of antibody concentration and preparation of 1 mg/mL working stocks The concentration of each of the test antibodies was measured twice using the Denovix spectrophotometer and the average of the two measurements were used in subsequent dilutions and experiments (Table 7). Furthermore, 1 mg/mL working stocks were prepared and measured twice on the Denovix spectrophotometer. The average of the two measurements was used in subsequent experiments (Table 8).

Table 7. Measured protein concentrations.

Table 8. Dilution and measurement of working stock protein concentrations.

Non-reducing and reducing SDS-PAGE analysis

The integrity of the test antibodies was analyzed by non-reducing and reducing SDS-PAGE. The antibodies were diluted according to Table 9 before loaded on the gel. The result showed that all antibodies migrated according to their expected molecular weights in non-reducing SDS-PAGE (Figure 1A). For NNV024 and NNV031 a faint band of unknown origin between 70-100 kDa was observed. Under reducing conditions, all antibodies separated into distinct bands corresponding to the heavy and light chains without any visual impurities (Figure 1 B). The SDS-PAGE also revealed highly similar bands for all antibodies, indicating that the protein concentrations had been accurately determined.

Coating levels of antibody variants Next, the antibodies were coated in ELISA wells (1000 - 0.45 ng/mL). To quantify the coating efficacy, two polyclonal detection antibodies, specific for either the human IgG heavy chain or the human kappa light chain were used. As shown in Figure 2A-D, both detection antibodies bound equally well to all the antibodies indicating uniform coating levels. Binding of antibodies to human FcRn at acidic and neutral pH in ELISA

Binding of the antibodies (1000 - 0.45 ng/mL) to human FcRn was performed at both acidic and neutral pH to mimic the endosomal and extracellular milieu. Under acidic conditions (pH 6.0), NNV029, NNV031 and NNV032 containing the REW substitutions showed improved binding to FcRn over the WT variants NNV023, NNV025, NNV024 and NNV030 (Figure 3A-B). In comparison, obinutuzumab and DuoHexabody-CD37 bound weaker than the WT NNV variants. The EC50 values for binding of the antibodies to FcRn at pH 6.0 are shown in Table 10. Under neutral pH conditions (pH 7.4), the REW containing NNV029, NNV030 and NNV032 bound FcRn weakly at the highest antibody concentration used, while the WT variants including obinutuzumab and DuoHexabody-CD37 showed even weaker or no binding (Figure 3C-D). The binding data also showed that afucosylation at the N297 position of the Fc (NNV024 and NNV032) or deletion of the C-terminal lysine in the IgG heavy chain (NNV030 and NNV031) did not affect pH-dependent FcRn binding.

Table 10. EC50 values for binding of test antibodies to FcRn at pH 6.0 (ng/mL).

FcRn binding kinetics of the antibodies at acidic pH in SPR

Next, the binding kinetics of the antibodies (1000 - 7.91 nM or 125 - 0.97 nM) to human FcRn at acidic pH (pH 6.0) were measured by SPR. In addition, relative binding responses were measured at neutral pH (7.4). The analysis showed that NNV029, NNV031 and NNV032 containing the REW substitutions bound reversibly to FcRn at acidic pH (6.0) with a faster on-rate and slower off-rate compared to the WT NNV variants as well as obinutuzumab and DuoHexabody-CD37 (Figure 4). This translated into a 40-fold lower KD for NNV029 and a 38.2 and 36.4-fold lower KD for NNV031 and NNV032, respectively (Table 11 ). No or minor binding responses between the antibodies and FcRn were observed under neutral pH conditions (Figure 5).

Table 11. FcRn binding kinetics to lgG1 variants at pH 6.0.

Binding of the antibodies to FcyRs in ELISA

All the antibodies bound the human low affinity FcyRs, including the allotypic variants of FcyRllla (V158 and F158) and FcyRlla (H131 and R131). The afucosylated variants NNV024 and NNV032 showed improved binding to both allelic variants of FcyRllla (Figure 6A-B) as well as FcyRlllb (Figure 6C). NNV025, NNV023, NNV029, NNV030 and NNV031 as well as obinutuzumab bound equally well to FcyRllla-V158, while obinutuzumab showed somewhat stronger binding to FcyRllla- F158. DuoHexabody-CD37 bound weaker to both FcyRllla allelic variants and FcyRlllb. All NNV variants bound equally well to FcyRlla (H131 and R131), FcyRllb and FcyRI, while reduced binding was observed for obinutuzumab and DuoHexabody-CD37 (Figure 6D-F and Figure 7A-B). The EC50 values for ELISA binding of the antibodies to the FcyRs are shown in Table 12 and Table 13.

Table 13. EC50 values for ELISA binding of test antibodies to Fc/RI (ng/mL)

Binding of the antibodies to human C1q Binding between the antibodies and human C1q was measured by coating high levels of the antibody variants (20.000 - 156.25 ng/mL) in ELISA wells. The results showed that NNV variants with the REW substitutions, NNV029, NNV031 and NNV032, as well as DuoHexabody-CD37 bound stronger to C1q compared to WT NNV variants NNV023, NNV025, NNV024 and NNV030 (Figure 8A-B). Only weak binding was observed for obinutuzumab. The control antibody N IP-IgG 1 - WT bound C1q while the NIP-lgG1-P329A C1q non-binding variant did not (Figure 8C).

DISCUSSION

Taken together, the concentration measurements, SDS-PAGE analysis and detection of coating efficiency in ELISA showed that all the antibodies were of high quality and purity.

For FcRn, improved pH-dependent binding was observed for the NNV variants containing the REW substitutions in ELISA. Only weak binding responses were detected at neutral pH. Afucosylation or deletion of the C-terminal lysine did not influence the binding properties of the NNV variants to FcRn in ELISA. Determination of the FcRn binding kinetics in SPR showed strongly enhanced binding for the REW containing NNV variants at acidic pH. The kinetic binding data revealed somewhat weaker binding when the REW substitutions were combined with removal of the C- terminal lysine or afucosylation. No or minor binding responses were detected at neutral pH in SPR.

As such, the FcRn binding data support that the NNV029, NNV031 and NNV032 variants will be rescued more efficiently from intracellular degradation by FcRn, and result in extended plasma half-lives in vivo compared to the other antibodies.

Furthermore, binding to classical FcyRs and C1q were shown to be influenced by the different alterations made to the antibodies. The afucosylated NNV variants (NNV024 and NNV032) clearly showed increased binding to both allelic variants of FcyRllla as well as FcyRlllb. However, the REW substitutions or removal of the C-terminal lysine did not have any additive effect in addition to afucosylation for binding to FcyRllla or FcyRlllb (NNV029 and NNV031 ). The results suggest that the afucosylated NNV variants may lead to increased ADCC activity against target cells. The different modifications made to the NNV variants did not affect their binding properties to any of the remaining FcyRs, including FcyRlla and FcyRI that mediate ADCP. For C1q, improved binding was observed for the REW containing NNV variants (NNV029, NNV031 and NNV032), which was on par with that of DuoHexabody-CD37. Afucosylation or removal of the C-terminal lysine did not affect the C1q binding properties of the antibodies in this assay (NNV024 and NNV030). The results suggest that NNV029, NNV031 and NNV032 may lead to enhanced CDC activity against target cells.

Since the antibodies were coated directly in ELISA and therefore randomly absorbed to the plastic, their orientation or structural flexibility may differ depending on their biophysical properties. This was particularly evident for obinutuzumab, which showed reduced binding to FcRn and almost all the effector molecules tested in ELISA. In contrast, the affinity of obinutuzumab for FcRn measured in SPR was on par with that of the WT NNV variants and DuoHexabody-CD37. The same trend was observed for DuoHexabody-CD37 in regards to FcRn and FcyR binding. Further, obinutuzumab showed only weak binding to C1q in ELISA.

From the obtained binding data, NNV032 with the REW substitutions and low fucose in its N297- linked glycan appears to be the best candidate to extend plasma half-life and potentiate elimination of target cells. However, testing in cellular and in vivo efficacy assay are needed to address this.

EXAMPLE 3 - Comparison of ADCC induced by NNV023, NNV024, NNV029, NNV030, NNV031, NNV032, duohexabody-CD37 or obinutuzumab in Ramos cells

The aim of this example is to compare induction of ADCC by NNV023, NNV024, NNV029, NNV030, NNV031 , NNV032, duohexabody-CD37 or obinutuzumab in Ramos cells

METHODS

Culturing and preparation of target cells

The Ramos cell line was cultured in RPMI1640 supplemented with GlutamaxX (Gibco, Paisley, UK), 10 % heat-inactivated FCS (Gibco) and 1% penicillin-streptomycin mix (Gibco). The incubation takes place in a humidified atmosphere with 5% CO2 at 37°C. Cell suspensions are diluted 1 :5 with pre-warmed medium twice a week (unless otherwise stated based on cell viability). To ensure an exponential growth at the beginning of the experiment, the cells were diluted 2 days before.

Experiment procedure

The target cells (Ramos) were diluted two days prior the day of the experiment. They were harvested and plated into white flat-bottom 96 well plates at concentration of approx. 25 000 cells/well in the 25 pL of the assay buffer/well.

The dilutions of the test items were prepared in triplicates at four different concentrations: 0.001 , 0.01 , 0.1 , and 1.0 pg/mL. The dilutions were prepared in 1 ,5 mL Eppendorf tubes (10-fold dilution) and then transferred into a v-bottom transparent 96 well plate following the layout described in the protocol (NNV020-p-076-2020.08). The solutions from this plate were added to both plates containing the target cells.

The cells and target Abs were co-incubated on the bench (LAF-bench) for 30 min. The effector cells from Promega ADCC Reporter Bioassay Core kit (Ref.G701A) were thawed, diluted and added to the assay plates with the target cells. The ratio of the Effector to Target cells became 2,65:1. After addition of the effector cells, the assay plates were incubated for 5 hours at 37°C in a humidified incubator.

After 5 hours of incubation, the cells were placed on the bench for 15 min to equilibrate the temperature to RT. Bio-Gio luciferase assay reagent was added to the plates (all wells containing cells plus the cells for background control that does not contain any Abs). Bio-Gio luciferase was added to these wells at the same time as to the other wells and incubated in the dark at RT for 10 or 15 min. The luminescence of the luciferin was measured using Tecan plate reader (integration time of the acquisition was 0,5 sec/well).

Data analysis

The value of the background control (the mean of RLU for cells without Abs) was withdrawn from the signals of all wells of the same plate. The mean RLU and standard deviation of triplicates for each concentration of the test antibody were calculated. The resulting data set was plotted using dot-line plot and clustered bar plot in the axes: RLU vs. [Ab] pg/mL. A degree of ADCC induction was also calculated relatively to Obinutuzumab for all other test items of the same concentration levels.

RESULTS

The result obtained from a single experiment testing ADCC activation in Ramos cells is presented in Figure 9.

The humanized antibodies (NNV023, NNV024, NNV029, NNV030, NNV031 , and NNV032) tested in this example showed a prominent induction of ADCC in Ramos cells.

The ADCC induction of NNV032 was similar to the one of NNV024 at the lower concentrations but reduced to the level of Obinutuzumab at higher concentrations 0,1 pg/mL and 1 pg/mL (100% and 99% of Obinutuzumab’s effect correspondingly).

The fully fucosylated NNV antibodies tested (NNV023, NNV029, NNV030, NNV031 ) showed a modest ADCC activation compared to Obinutuzumab with NNV023 being the most efficient: 40% at 0,001 pg/mL and 76,7% at 0,1 pg/mL (all relatively to ADCC of Obinutuzumab of the same concentration). NNV029 showed the second high ADCC activation of all NNVs with normal N- glycan tested followed by NNV031 (min: 37,7% at 0,001 pg/mL; max: 67% at 0,1 pg/mL). ADCC induced by NNV030 was the lowest of all NNV Ab variants tested (min: 29.7% at 0,001 pg/mL; max: at 59.7% at 0.1 pg/mL).

Duohexabody-CD37 induced the lowest ADCC in Ramos cells, from 16.3% at 0.001 pg/mL to maximum of 32.7% of Obinutuzumab at 0.01 pg/mL. The further increase in concentration of duohexabody-CD37 reduces induction of ADCC to only 16.3% at 1.0 pg/mL. This effect may presumably be attributed to a spontaneous hexamerisation of lgG1 at higher concentrations. ADCC induced by NNV032 and NNV024 at 0.1 pg/mL was 4.2-fold and 3.6-fold times higher than that of duohexabody-CD37. At the same concentration level, ADCC induced by fully fucosylated Abs (NNV023, NNV029, NNV030, NNV031 ) was 2.8-2.2-fold times higher than of duohexabody- CD37.

DISCUSSION

In order to assess the effect of REW point mutations introduced to the Fc part of humanized Abs, the following pairs of antibodies should be compared: NNV029 (REW) vs. NNV023 (WT), and NNV032 (afucsoylated + REW- termLys) vs. NNV024 (afucosylated).

Comparison of NNV023 and NNV029 displays only a very limited decrease of the ADCC when REW mutations added to an antibody with fully fucosylated N-glycan. For the lowest concentrations tested (0.001 and 0.01 pg/mL) the ADCC effects of both NNV023 and NNV029 were identical. A slightly lower (10%) ADCC was apparent for NNV029 compared to NNV023 only at 0.1 pg/mL. At 1 .0 pg/mL the ADCC values were overlapping.

The effect of REW was more pronounced if the Ab had an afucosylated N-glycan. The difference between NNV024 and NNV032 was not pronounced at the lowest concentrations tested (0.001 and 0.01 pg/mL), but the REW mutations somewhat reduced the ADCC effect of afucosylation 0.1 pg/mL (13.3 %) and 1.0 pg/mL (10.5 %).

It seems that the absence of fucose in the core of the N-glycan is the key factor enhancing ADCC induction. Antibodies manufactured under different conditions (e.g. different technology of afucosylation and diff. manufacturers, different manufacturing batches of cells or even different cell lines) may have different composition of the N-glycan or different fraction of afucosylated N-glycan in the batch. The three afucosylated Abs tested in the example were manufactured at different dates or by different manufacturers: NNV024 and NNV032 both incorporating GlymaxX technology but manufactured in 2020 and 2021 correspondingly at Evitria AG, and Obinutuzumab manufactured using GlycArt technology and manufactured by Roche. Manufacturing process and QC of Obinutuzumab is well established and controlled, which a transient transfection and expression was used for manufacturing of NNV024 and NNV032. Therefore, the performance of these Abs may vary from batch to batch of NNV variants compared to each other and relatively Obinutuzumab. Qualitative and quantitative analysis of the glycan content of GlymaxXO-carrying Abs (NNV024 and NNV032) and obinutuzumab is warranted in order to understand the observed differences in ADCC for these Abs.

In order to assess the effect of removal of c-terminal lysine the following pairs of Abs should be compared: NNV023 (WT) with NNV030 (without c-terminal lysine); NNV029 (REW) and NNV031 (REW, without c-terminal lysine).

When comparing ADCC of NNV023 with its homolog without C-terminal lysine, NNV030, an unexpected drop of ADCC activation was found for the latter Ab. The drop was found for majority of concentrations tested. The largest difference was observed for 0.1 and 1.0 pg/mL (reaching 20% drop for NNV030 relatively to NNV023 at 1.0 pg/mL). The mechanism for this decrease in the ability to induce ADCC for WT Ab lacking c-terminal lysine is unknown. These results are based on only one experiment.

The deletion of c-terminal Lysine from a REW-harbouring Ab such as NNV029 does not change its ability to induce ADCC. Indeed, the difference in ADCC between NNV029 and NNV031 lays within margin of standard deviation.

The induction of ADCC by NNV032 was similar to the one of NNV024 at the lower concentrations tested (0.001 - 0.01 pg/mL), but was reduced to the level of obinutuzumab at concentrations 0.1 - 1.0 pg/mL.

The antibodies with non-modified N-glycan (NNV023, NNV029, NNV030, NNV031 ) showed a modest ADCC activation compared to obinutuzumab with NNV023 being the most efficient. The removal of lysine from c-terminus of heavy chain does not affect ADCC of REW-mutated antibodies, but an unexpected drop of ADCC (20%) was observed for NNV030, without c-terminal lysine, compared to NNV023 (WT).

Duohexabody-CD37 induced the lowest ADCC in Ramos cells of all antibodies tested.

EXAMPLE 4 - Comparison ofADCP induced by NNV023, NNV024, NNV029, NNV030, NNV031, NNV032, duohexabody-CD37 and obinutuzumab in Ramos cells

The aim of this example is to compare induction of ADCP by NNV023, NNV024, NNV029, NNV030, NNV031 , NNV032, duohexabody-CD37 and obinutuzumab in Ramos cells.

METHODS

Three Promega ADCP reporter bioluminescence bioassays for quantifying biological activity of therapeutic antibodies on pathway activation of ADCP were used in this example. The assays are designed to test different activation pathways of ADCP: binding to either FcyRlla-H131 , FcyRlla- R131 , or FcyRI.

Except for rituximab and obinutuzumab, all antibodies used in this example, were manufactured as described in example 1.

Culturing and preparation of target cells

All reagents for the cell culturing are purchased from Gibco. The cell lines are cultured in RPMI- 1640 supplemented with GlutamaxX, heat-inactivated FCS (10 % v/v) and penicillin-streptomycin mix (1% v/v). The incubation takes place in a humidified atmosphere with 5% CO2 at 37°C. Daudi are usually diluted 1 :5 with pre-warmed medium twice a week (unless otherwise stated, based on current cell viability). Prior any experimental use, the cells should be given min. 1-1 ,5 week to recover after thawing. The cells will be diluted 2-3 days before start of the experiment to ensure an exponential growth at the beginning of the experiment.

Experiment procedure

The target cells (Ramos) were diluted two days prior the day of the experiment. They were harvested and plated into the four white flat-bottom 96 well plates at concentration of approx.

25 000 cells/well in the 25 pL of the assay buffer/well.

The dilutions of the test items were prepared in triplicates at four different concentrations: 0.01 , 0.1 , 1.0, and 10.0 pg/mL. The dilutions were prepared in 1.5 mL Eppendorf tubes (10-fold dilution) and then transferred into a v-bottom transparent 96 well plate following the layout described in the protocol (NNV020-p-076-2020.09). This plate was used as a template. The dilutions from this plate were dispensed (25 pL/well), retaining the layout, to the three plates containing the target (Daudi) cells. The fourth plate with the cells was left with no Abs added.

The cells and the test Abs were co-incubated on the bench (LAF-bench) for 15 min. The fourth plate was treated in the same manner.

After 15 min, the vials of the effector cells from the Promega ADCP Reporter Bioassays Core kits ( FcyRlla-H131/-R131 and FcyRI ) were thawed, diluted and added to the assay plates with the target cells (25 pL of the diluted in the assay medium effector cells/well). The ratio of the Effector to Target cells was estimated as 2.65:1. The blank wells contained target and effector cells, but not the test antibodies.

After addition of the effector cells, the assay plates were incubated for 18 hours at 37°C in a humidified incubator (5% CO2).

After 18 hours of incubation, the cells were placed on the bench for 15 min to equilibrate the temperature to RT. The premixed, in accordance with the instructions of the kit’s manual, Bio-Gio luciferase assay reagent (75 pL/well) was added to the plates. After addition of Bio-Gio luciferase, the system was incubated in the dark at RT for 10 min. The luminescence of the luciferin was measured using Tecan plate reader on 10 and 15 min after adding Bio-Gio reagent. Integration time of the acquisition was set to 0.5 sec/well, temperature of the instrument chamber was set to 21 °C.

Data analysis

The negative control values (mean RLU of the wells with T&E cells, but without Abs) was subtracted from the signals of all wells of the corresponding assay type.

Mean RLU and standard deviation (SD) of triplicates of the test antibody at each concentration level were calculated and plotted using dot-line plot and the clustered bar plot.

In addition, a relative ADCP induction vs. obinutuzumab was calculated for the test items at the corresponding concentration levels.

RESULTS

The effect of REW on the interaction with FcyRlla-H131 receptors

Duohexabody-CD37 had slightly higher response than NNV029, NNV031 and NNV032 at 0.1 pg/mL, but it was reversed at 1 pg/mL and the NNV antibodies with REW mutations had a significantly higher response than duohexabody-CD37 at 10 pg/mL (Figure 10).

Rituximab showed very good binding to FcyRlla-H131 compared to NNV023 and NNV024 displaying almost linear dose-dependence on the concentrations 0.01 - 1.0 pg/mL Around 1.0 pg/mL rituximab reaches saturation and the result of RTX at 10.0 pg/mL is the same as for 1.0 pg/mL.

Unlike rituximab, the NNV029, NNV031 , and NNV032 demonstrate solid linear response on the whole range of tested concentrations (0.01 - 10.0 pg/mL) (Figure 10). The dose-response of NNV029 and NNV031 are overlapping and are steeper than the one of NNV032. The doseresponse profile of NNV032 is very similar to of Rituximab on the ranges where Rituximab displays the linear growth. At the concentration of 10 pg/mL NNV032 is approximately 1.5 times more potent than Rituximab or Obinutuzumab.

The effect of REW on the interaction with FcyRlla-R131 receptors

The effect of REW mutations on inducing ADCP-related signalling via FcyRlla-R131 is even stronger than for the H131 variant (Figure 31). The Abs harboring REW mutations (NNV029, NNV031 , NNV032) showed absolute superiority in inducing ADCP response via FcyRlla-R131. NNV032 showed the highest response. NNV029, NNV031 and NNV032 displayed a steep dosedependent growth through all concentrations tested.

Obinutuzumab also induces a strong response via this receptor, while rituximab showed a result which was only slightly better than for the NNV antibodies without REW mutations, NNV023 and NNV024. However, as for rituximab for FcyRlla-H131 , obinutuzumab reaches saturation for concentrations at or above 1.0 pg/mL.

The effect of REW on the interaction with high affinity FcyRI receptors

All antibodies tested had similar response in the FcyRI assay, except for duohexabody-CD37 which had a significantly lower response (Figure 12). NNV023 and NNV024 display approximately 90-95% ADCP activation as compared to obinutuzumab, while the REW Abs have only 80-85 % of the activity of obintuzumab in this assay.

DISCUSSION

The induction of ADCP (FcyRlla- H131/R131) was greatly improved after REW mutations were introduced into the Fc part of such antibodies as NNV029, NNV031 , and NNV032 as compared to their predecessors without REW. On the other hand, the ADCP activation via FcyRI receptor was reduced by 12%, in average for all REW Abs, compared to the predecessor without REW.

A limited modulation of ADCP induction is observed when afucosylated N-glycan co-exists with REW mutations. For FcyRlla-H131 variant afucosylation reduces 1.15-fold times the effect of REW mutations on ADCP, while it increases the efficiency of ADCP induction 1.11 -fold times when FcyRlla-R131 is recruited.

There are no obvious signs indicating that removal of c-terminal lysine from Abs impacts induction of ADCP-related signalling. The overlapping dose-response profiles of NNV023 (with c-terminal Lys) and NNV030 (no c-terminal Lys) as well as NNV029 (with REW and c-terminal Lys) and NNV031 (with REW and no c-terminal Lys) are indicative that removal of c-terminal lysine from the Fc part of antibody do not affect ADCP signalling.

The induction of ADCP was greatly improved after introduction of REW mutations in the Fc part of NNV antibodies. The induction of ADCP was superior for NNV029, NNV031 and NNV032 as compared with obinutuzumab, rituximab and duohexabody-CD37. EXAMPLE 5 Solution phase complement activation of humanized anti-CD37 antibody variants Activation of the complement cascade by IgG antibodies via the classical pathway requires antigen binding and on-target hexamerization of the antibody at the cell surface. The ability of IgG antibodies to form such hexamers can be enhanced by engineering of the Fc. This is exemplified by the Hexabody technology, where the E430G substitution strengthens intermolecular Fc:Fc contacts and enhances the ability of the antibody to form hexamers that trigger complement dependent cytotoxicity (CDC). However, it is crucial that such engineered antibodies only trigger complement activation when bound to their cognate antigen to avoid unspecific activation and adverse effects. This is called on-target complement activation.

Therefore, the aim of the present example is to measure potential off-target complement activity of engineered humanized anti-CD37 lgG1 antibody variants (NNV) side-by-side with commercially obtained obinutuzumab (anti-CD20; lgG1 , Gazyvaro) and a recombinant form of DuoHexabody- CD37 containing the E430G substitution (anti-CD37; Biparatopic lgG1 ; DuoHexabody-CD37) in normal human serum (NHS).

The antibodies (200 pg/mL) were incubated in NHS at 37°C for 1 hour in the absence of antigen. Potential off-target complement activation was determined by measuring the serum concentration of C4d, a soluble biproduct of the complement cascade, using ELISA. Furthermore, the concentration of higher order IgG assemblies (complexes) in serum was measured by ELISA. As negative controls, NHS only, as well as two anti-5-iodo-4-hydroxy-3-nitrophenacetyl (NIP) antibodies are used (NIP-lgG1-WT and NIP-lgG1-P329A (the P329A mutation makes the antibody C1q non-binding). As a positive control, NIP-lgG1-E345R/E430G/S440Y (RGY), which is known to form hexamers in solution in the absence of antigen and activate the complement cascade is used. The IgG 1 antibody variants tested are shown in Table 14.

Table 14. The antibodies used in this example.

METHODS

Solution-phase complement activation and IC formation

The antibodies were added to 0.5 mL NHS to a final concentration of 200 pg/mL and incubated for 1 hour at 37°C. NIP hapten specific lgG1-WT, lgG1-P329A and NHS only were used as negative controls, while NIP hapten specific lgG1-RGY was used as a positive control. The resulting concentration of C4d or higher order IgG assemblies were determined using the MicroVue C4d EIA and MicroVue CIC EIA kits, following the manufacturer’s instructions. Illustrations of the EIA kits are shown in Figure 13. The signals obtained for the test antibodies in NHS were interpolated in Graphpad Prism to a standard curve supplied in the kits.

RESULTS

The results showed that of the antibodies tested, only the NIP-lgG1-RGY variant resulted in any increase in NHS C4d concentration (Figure 14A). The C4d levels detected in NHS incubated with the remaining IgG 1 variants were similar and not significantly different from the levels detected in NHS only or in NHS incubated with NIP-lgG1-WT or NIP-lgG1-P329A. In line with this, only the NIP-lgG1-RGY variant increased the concentration of IgG complexes, while the remaining IgG variants and control antibodies, again, were comparable to the levels detected in NHS only (Figure 14B).

DISCUSSION

To be used safely, antibodies engineered for enhanced hexamer formation and CDC activity must only be active when bound to their target antigen. Off-target activation of the complement cascade may lead to adverse effects, as well as faster clearance of the drug. DuoHexabody-CD37 which takes advantage of the hexabody technology, did not lead to off-target complement activation in our experiments. However, NIP-lgG1-RGY, containing two additional amino acid substitutions, which further strengthen Fc:Fc contacts, leads to elevated C4d levels and formation of IgG complexes in NHS, independently of antigen binding. Importantly, none of the engineered NNV anti-CD37 lgG1 variants, including those with the REW substitutions, lacking the C-terminal lysine or afucosylation, resulted in any off-target complement activation at the concentration tested.

Neither of the engineered anti-CD37 lgG1 variants (NNV) activated the complement cascade in an antigen independent manner in NHS at concentrations at or below 200 pg/mL. As such, no adverse effects or increased clearance due to off-target hexamerization or complement activation are expected.

EXAMPLE 6 Differential scanning fluorimetry (DSF) to determine Tm (°C) and aggregation onset of humanized anti-CD37 antibody variants.

The aim of the experiment was to determine the Tm (°C) and aggregation propensity of engineered anti-CD37 lgG1 variants (NNV) side-by side with commercially obtained obinutuzumab (anti-CD20; lgG1 ; Gazyvaro) and a recombinant form of DuoHexabody-CD37 (anti-CD37; Biparatopic lgG1 ; DuoHexabody-CD37) using label-free fluorometric analysis (Tm) and dynamic light scattering (DLS), respectively.

Label-free fluorometric analysis (nanoDSF) is based on the fluorescent amino acid tryptophan, generally located in the hydrophobic core of proteins, shielded from the surrounding aqueous solvent. Upon unfolding, tryptophan is exposed, which alters its photophysical properties. By detecting changes in tryptophan fluorescence intensity and its emission peak shift, the transition of a given protein from the folded to the unfolded state can be recapitulated. This way, the Tm (°C) can be determined. The Prometheus NT.48 instrument monitors the shift in intrinsic tryptophan fluorescence of proteins upon unfolding by detecting the fluorescence at emission wavelengths of 330 and 350 nm. The protein Tm, where half of the protein is folded and the other half is unfolded, is determined from the 1st derivative of the ratio between the fluorescence intensities (350/330 nm). nanoDSF is routinely used for monitoring protein purification, stability during formulation, developability, production and manufacturability of biologies.

DLS is based on intensity fluctuations of laser light scattered by molecules/particles moving in Brownian motion. The diffusion coefficient is determined and converted to particle size via the Stokes-Einstein equation. DLS can determine the hydrodynamic size of protein monomers and small aggregates in the nanometer range. Prometheus NT.48 uses DLS to determine aggregation status by measuring loss of light intensity due to scattering. It is routinely used for screening recombinant proteins expressed in different systems, biologies formulation screening, sample optimalization for biophysical assays and characterization of self-interactions. METHODS

The test antibodies used in this example are shown in Table 15.

Table 15. Test antibodies

Nanoscale differential scanning fluohmetry (nanoDSF)

The antibodies were diluted to 1 mg/mL in 1xPBS (without CaCl2 or MgClz) before drawn into capillaries. The Prometheus NT. 48 nanoDSF instrument was loaded with the capillaries containing the antibodies. Each antibody variant was added in triplicates. The instrument was set to gradually increase the temperature from 25 °C to 95 °C (1°C/min). The ratio between 350 and 330 nm wavelengths (350/330 nm) was continuously monitored and plotted against temperature (°C). The melting temperature Tm (°C), in which 50% of the protein is unfolded, was determined by the first derivative of the 350/330 nm ratio, using the Prometheus ThermoControl software. The 1^st derivative of scattering causing loss of excitation light intensity was plotted against the temperature range tested. The increase in scattering through the temperature range marks the onset of protein aggregation and can be used to predict the aggregation propensity of the antibodies tested.

RESULTS

The analysis showed that obinutuzumab had the highest T m (°C) (81.3 °C) of the antibody variants (Figure 15, Table 16). The NNV variants had somewhat lower Tm (°C) (74.7-76.0 °C), 5.3-6.9 °C lower than obinutuzumab. DuoHexabody-CD37 had the lowest Tm (°C) (59.1 °C), which was 22.2 °C lower than obinutuzumab and 15.6-16.9 °C lower than the NNV variants.

In line with the thermal stability data, obinutuzumab was most resistant to aggregation through the increasing temperature range, with a scattering peak reached at 81.9 °C (Figure 16, Table 16). Of the NNV variants, NNV025 was most resistant to aggregation, with a scattering peak at 77.8 °C.

The remaining WT NNV variants showed scattering peaks at slightly lower temperatures (76.7-76.8 °C), while the NNV variants containing the REW substitutions (NNV029, NNV031 and NNV032) showed scattering peaks between 76.0 and 76.2 °C.

Table 16. Tm (°C) and peak scattering temperatures

DISCUSSION

Therapeutic antibodies should meet a standard of criteria regarding the feasibility of their manufacture and biophysical stability. For example, heat induced unfolding generally leads to irreversible conformational changes impairing their function. The Tm of late-stage clinical development and approved monoclonal antibodies, in which 50% of the protein is unfolded, has been found to be evenly distributed between 60-85 °C (1). Furthermore, the aggregation propensity of monoclonal antibodies may affect both their function and shelf-life. These two parameters can be used as an indication of whether the developability profile of a given monoclonal antibody is favorable or not.

The NNV variants tested showed a Tm (°C) between 74.7 and 76.0 °C. Furthermore, the temperature at which the NNV variants aggregated, was only 1-1.8 °C lower than their Tm, suggesting that the antibodies are not particularly prone to self-association. The results further reveled that afucosylation, removal of the C-terminal lysine or addition of the REW substitutions did not result in any large fluctuations in thermal stability or heat induced aggregation propensity.

Based on these results, no negative impact on downstream production, labeling or biophysical stability of the engineered anti-CD37 NNV variants are expected.

EXAMPLE 7 Human endothelial recycling assay (HERA) to determine FcRn mediated cellular transport of humanized anti-CD37 antibody variants

The prevalent antibody class in blood, IgG, has an average plasma half-life of 20-23 days. The long half-life of IgG is due to its molecular size (146 kDa) above the renal clearance threshold and its interaction with the neonatal Fc receptor (FcRn). FcRn is a major histocompatibility class I (MHCI) related molecule consisting of a transmembrane heavy chain that non-covalently associates with p2-microglobulin. FcRn binds in the CH2-CH3 elbow region of the IgG Fc in a strictly pH-dependent manner, with binding at acidic (pH 6.0) and no binding or release at neutral pH (7.4). FcRn is predominantly located within acidified endosomes, where the low pH allows binding of IgG taken up by fluid phase pinocytosis. FcRn then recycles its IgG ligand back to the cell surface for release into circulation upon exposure to the neutral pH of the blood. Proteins or antibodies that do not bind the receptor are sorted into lysosomes and degraded.

Development of engineered IgG molecules with improved pharmacokinetics requires efficient screening methods in which FcRn mediated cellular transport can be quantitatively monitored. HERA - human endothelial cell recycling assay, is here used to quantify FcRn mediated cellular transport of engineered humanized anti-CD37 lgG1 variants (NNV) side-by-side with commercially obtained obinutuzumab (anti-CD20; lgG1 , Gazyvaro) and a recombinant form of DuoHexabody- CD37 (anti-CD37; Biparatopic lgG1 ; DuaHexabody-CD37).

The human endothelial cell line HMEC-1-HA-hFcRn-EGFP (HMEC1), overexpressing FcRn, was used to measure cellular uptake, FcRn-mediated recycling and intracellular retention (residual amount) of the antibodies. The amount of antibody present in each sample fraction was then determined by ELISA. Finally, a HERA score was determined based on recycled and residual amounts. A high HERA score is indicative of extended plasma half-life of a given antibody variant in a pre-clinical hemizygous human FcRn transgenic mice. METHODS

The antibodies (see Table 17) were stored at 4°C. NIP hapten (4-hydroxy-5-iodo-3- nitrophenylacetyl) specific lgG1-WT and lgG1-REW with known transport properties was included as controls.

Table 17. Test Antibodies.

Cell culture

HMEC1-HA-hFcRn-EGFP (HMEC1) cells were maintained in MCDB 131 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 25 pg/mL streptomycin, 25 U/mL penicillin, 10 ng/mL mouse epidermal growth factor (mEGFR), 1 pg/mL hydrocortisone, 5 pg/mL blasticidine S-HCI and 100 pg/mL G418. The cells were grown in a humidified 37 °C/5% CO2 incubator.

Human endothelial recycling assay (HERA)

1. 7.5 x 10⁴ HMEC1 cells stably expressing HA-hFcRn-EGFP were seeded per well in two 48-well plates and cultured for one day in complete growth medium as specified above. Prior to experiments, the cells were washed twice and starved for 1 hour in Hank's Balanced Salt Solution (HBSS).

2. 800 nM of each antibody, in 250 |_iL HBSS (pH 7.4), was added in triplicates to plate 1 and plate 2. Plate 1 was used to measure cellular uptake, while plate 2 was used to measure recycling and residual intracellular amounts. An overview of the assay is shown in Figure 17 and the plate layout is shown below.

3. Following incubation for 3 hours at 37°C/5%, CO2, cells in plate 1 and 2 were washed 4 times with ice cold HBSS. Then plate 1 (uptake fraction) was placed at -80°C, while fresh growth medium (220 ju.L) (without blasticidine S-HCI and G418) with 1x non-essential amino acids (NEAA) were added to plate 2 and incubated for 3 hours at 37°C/5%, CO2.

4. Medium samples (220 pL) (recycled fraction) were collected from plate 2 before the cells were washed 4 times with ice cold HBSS and stored at -80°C until isolation of total cell lysate (residual amount fraction).

5. Total cell lysate from plate 1 (uptake fraction, Table 18) and plate 2 (residual amount fraction, Table 19) was isolated by addition of 220 |_iL RIPA buffer containing 1x Protease Inhibitor Cocktail and incubation on ice/shaker for 10 min. The plates were then centrifuged for 15 min at 10.000 x g at 4 °C before isolation of supernatant.

6. The amounts of antibody in the different sample fractions were quantified by ELISA as described in the next section.

Table 18 HERA plate 1. Uptake fraction:

Table 19 HERA plate 2. Recycling fraction and residual amount fraction:

Quantification of antibodies from HERA

1. 96 well EIA/RIA plates were coated with 100 |_iL of an anti-human IgG antibody from goat diluted to 1 pg/mL in 1xPBS and incubated overnight at 4°C.

2. Plates were washed four times with PBS containing 0.05% Tween20 (T) using a Hydrospeed™ plate washer (Tecan). 3. Remaining surface area was blocked with PBS/T containing 4% skimmed milk powder (S) for 1 hour at room temperature to prevent unspecific binding of molecules added in the remaining steps.

4. Plates were washed four times with PBS/T using a Hydrospeed™ plate washer.

5. HERA samples (1 :1 serial dilution in PBS/T/S) were added to the plates and incubated for 1 hour at RT.

6. Antibody captured from samples was detected by an alkaline phosphatase (AP) - conjugated polyclonal anti-human IgG (Fc specific) Ab from goat (diluted 1 :5000 in PBS/T/S) and visualized by addition of p-nitrophenylphosphate (1 mg/mL in diethanolamine buffer).

7. Plates were washed four times with PBS/T using a Hydrospeed™ plate washer.

8. Plates were developed for 20-30 min before the 405 nm absorption values were recorded using a TECAN Sunrise plate reader. The ELISA setup is shown in Figure 17.

RESULTS

The results showed that the NNV IgG 1 variants with the REW substitutions, namely NNV029, NNV031 and NNV032 were more efficiently taken up and recycled by FcRn in HMEC1 cells compared to the WT variants NNV025, NNV023, NNV024 and NNV030 (Figure 18A-B). Obinutuzumab and DuoHexabody-CD37 were recycled in amounts that were comparable to the NNV IgG 1 WT variants. Quantification of intracellular residual amounts at the assay endpoint showed that the REW containing NNV029, NNV031 and NNV032 were not retained inside the cell by FcRn in higher amounts than the WT antibodies (Figure 18C). The results obtained for the NNV IgG 1 variants were comparable to the behavior of the WT and REW NIP hapten specific control antibodies. Calculating the HERA score (based on dividing recycled amount by residual amount) resulted in higher scores for the REW containing variants NNV029, NNV031 and NNV032 compared to the WT NNV variants (Figure 19).

DISCUSSION

The results from the HERA assay showed that all antibodies were rescued from lysosomal degradation by FcRn in HMEC1 cells and released back into the extracellular environment. The more efficient internalization of NIP-lgG1-REW, NNV029, NNV031 and NNV032 may be a result of weak residual FcRn binding at neutral pH (Example 2). Importantly, the REW containing variants were also released back into the medium in higher amounts and were not retained inside the cells in higher amounts than the WT lgG1 variants. This means that the REW containing variants were efficiently released from FcRn back into the extracellular environment during exocytosis.

The higher HERA scores obtained for NIP-lgG1-REW, NNV029, NNV031 and NNV032 suggest that these antibodies will have extended plasma half-lives.

EXAMPLE 8 Comparison of CDC induced by NNV023, NNV024, NNV025, NNV029, NNV030, NNV031, NNV032, Duohexabody CD37, and Obinutuzumab in Raji and Daudi cells

AIM

The goal of this study was to measure the ability of the anti-CD37 antibodies NNV023, NNV024, NNV025, NNV029, NNV030, NNV031 and NNV032 to induce complement dependent cytotoxicity on the target cells Raji and Daudi. The CDC performance of these antibodies was evaluated through the quantification of cell viability upon treatment and compared to the CDC activity displayed by the anti-CD20 antibody Ofatumumab (positive control) and the C1q-binding impaired Ofatumumab P329A (negative control). Moreover, this study aimed to compare the CDC potential of NNV antibodies against the approved anti-CD20 therapeutic antibody obinutuzumab (Gazyvaro, Roche) and against Duohexabody-CD37 (HexaBody®-CD37 or GEN-3009, Genmab), a biparatopic anti-CD37 antibody currently in Phase l/ll of clinical testing.

MATERIALS AND METHODS

Table 20 presents an overview of the test items.

Table 20: Antibodies used in the study.

Human Complement Serum (CTS-006) from Creative Biolabs was used as a source of complement. The C3&C5 removed Normal Human Serum, which has been deprived of essential proteins of the complement cascade, served as a negative control to rule out the complement dependency of antibody-induced cytotoxicity.

Culturing of target cells

The cell lines Raji and Daudi were cultured in RPMI1640 supplemented with GlutamaxX (Gibco, Paisley, UK), 10% heat-inactivated FCS (Gibco) and 1% Penicillin-streptomycin mix (Gibco). Cells were incubated in a humidified atmosphere with 5% CO2 at 37°C. The cell suspensions were usually diluted to a concentration of 0,4 million cells/mL with pre-warmed medium twice a week. Three days before the start of the CDC experiment, both Raji and Daudi cell lines were diluted to ensure that they were in the exponential phase of growth at the beginning of the experiment.

Experiment procedure in short

On the day of the experiment, the target cells Raji and Daudi were harvested, washed and seeded into two black flat-bottom 96 well plates (CDC plates) at a concentration of approx. 1 ,5 million cells/ml (37500 cells/well) in 25 pL of serum-free RPMI.

Serial dilutions of the test items (1 :25) were prepared in duplicates at three different concentrations: 0,016, 0,4 and 10 pg/mL. In addition, a control solution containing no antibody was prepared. 25 pL of each dilution were added to the CDC plates. To promote cell opsonization, target cells and test antibodies were incubated for 20 min at 37°C in a humidified incubator.

Afterwards, 50 pL of 25% Human Complement Serum (CTS-006, Creative Biolabs) were added to one of the CDC plates and 50 pL of C3&C5 Removed Normal Human Serum (CTS-054, Creative Biolabs) to the other one. The final serum concentration for the assay was 12,5%. The plates were incubated for two hours at 37°C in a humidified incubator to promote complement-dependent cell lysis.

To measure cytotoxicity, the cell viability reagent AlamarBlue was added to the plates (1 :10 dilution), in all cell-containing wells plus 8 background wells without any cells. Upon overnight incubation (18 hours, 37 °C, 5% CO2), the fluorescent signal derived from resazurin reduction was acquired using the Ascent Fluoroskan fluorometer (excitation: 544nm; emission:590 nm).

Data analysis

The cell-specific viability signal was adjusted by dividing the fluorescence measured in each cellcontaining well on the average of the signals from background wells within the same plate (signal/noise ratio). For both experiments, the average and standard deviation of the duplicates for each concentration of all the test items were calculated. The resulting data set was plotted using a clustered bar plot, with the following axes: signal/noise ratio (AU) vs. Antibody concentration.

RESULTS

The results obtained from the combination of two CDC biological replicates in both Raji and Daudi cells are described in the next two sections.

CDC induction in Raji cells

The data shown in Figure 20 represent the CDC effects of the screened antibodies on the target cells Raji.

In 12,5% Human Serum Complement (Figure 20A), DuoHexabody-CD37 activates CDC prominently, as shown by a strong decrease of cell viability compared to the control (around 40% reduction at 10 pg/mL). The CDC effects of DuoHexabody-CD37 were appreciated already with the lowest concentration (0,016 pg/mL) and were in the same range as the positive control Ofatumumab WT with 0,4 pg/mL of antibody (relative cell viability of 69,1% for DuoHexabody- CD37 vs 70,6% for Ofatumumab WT). At the highest concentration, Ofatumumab WT works slightly better in reducing Raji cell viability (46,6% for Ofatumumab vs 60,6% for DuoHexabody- CD37).

In general, the humanized NNV antibodies tested in this study displayed very modest CDC activation in Raji cells. The best CDC activation was achieved by NNV024 and NNV032, two afucosylated antibodies. Both are able to reduce cell viability by around 11% at a concentration of 0,4 pg/mL (88,97% for NNV024 and 89,4% for NNV032), and by around 20% at the concentration of 10 pg/mL (80,1% for NNV024 and 83,4% for NNV032). Comparing these results with the CDC activation by the negative control Ofatumumab P329A, a small reduction (100% vs ca 89% NNV024 and NNV032) was observed with 0,4 pg/mL of antibody. At the highest concentration, both NNV024 and NNV032 seem to activate CDC at a minor extent than Ofatumumab P329A (73,5% relative cell viability). However, the CDC induction achieved by the negative control antibody might be due to residual C1q binding or cross-linking effects that occurred at high concentrations.

NNV023, NNV025 and NNV030 were the worst antibodies in the tested panel. At any concentration, they did not exhibit any complement-dependent cytotoxicity, leaving cell viability on similar values as in the control. NNV029 and NNV031 , two REW containing antibodies, had very limited effects (around 12-15% decrease of cell viability) only at the highest concentration.

The assessment of the CDC performance of the commercially approved Obinutuzumab revealed that also this antibody killed Raji cells in a limited manner. At the concentration of 0,4 pg/mL, almost no reduction of cell viability could be appreciated (98% vs control), while 10 pg/mL of Obinutuzumab activated CDC at rates similar to the ones observed for NNV024 and NNV032 (relative cell viability 82,5% for Obinutuzumab vs 80% for NNV024 and 83,4% for NNV032).

To confirm that the cytotoxic effects induced by the antibodies in the panel were dependent on the complement cascade, the assay was also performed in 12,5% human serum deprived of the complement proteins C3 and C5, essential for the formation of membrane attack complex. The results obtained are represented in the bar plots of Figure 20B. As expected, no relevant reduction of cell viability was observed with any of the antibodies tested, even at the highest concentration.

CDC induction in Daudi cells

The data shown in Figure 21 represent the CDC effects of the screened antibodies on the target cells Daudi.

In 12,5% Human Complement Serum, DuoHexabody-CD37 induces very strong cytotoxicity on Daudi cells. In particular, cell survival is reduced by around 90% in presence of both 0,4 pg/mL and 10 pg/mL of antibody (relative cell survival 13,3% at 0,4pg/mL and 12,5% at 10 pg/mL). CDC activation looks more prominent for DuoHexabody-CD37 than for the positive control Ofatumumab WT at the concentration 0,4 pg/mL (relative cell viability of 13,3% vs 54,8%), while both antibodies drastically reduce cell survival at 10 pg/mL (12,9% for Ofatumumab WT and 12,5% for DuoHexabody-CD37).

Among the humanized NNV articles tested, the REW-containing antibodies NNV029 and NNV031 are the best at inducing CDC. Following a similar trend, NNV029 and NNV031 were able to decrease cell viability by around 22 % at the concentration of 0,4 pg/mL (relative cell viability 79,5% for NNV029 and 77,6% for NNV031) and by 43% at 10 pg/mL (57,1% for NNV029 and 58% for NNV031).

The afucosylated antibodies NNV024 and NNV032 exhibited modest CDC effects on Daudi cells, by reducing cell survival of around 20-30% at both 0,4 and 10 pg/mL.

As observed in Raji cells, NNV023, NNV025 and NNV030 were the worst performing antibodies in the panel of tested antibodies and did not induce cytotoxicity at any concentration. The evaluation of the performance of Obinutuzumab in CDC revealed very limited cytotoxicity on Daudi cells, with values similar to the ones showed by the negative control Ofatumumab P329A. It was possible to appreciate a slight reduction of cell viability only at 10 pg/mL (relative cell viability 93%), and no effects at lower antibody concentrations. In contrast with the observations made in Raji cells, the humanized antibodies NNV024, NNV029, NNV031 and NNV032 achieved significantly better CDC levels than Obinutuzumab in Daudi cells.

As for Raji cells, the ability of the tested antibodies to kill Daudi cells in a complement dependent manner was tested in 12,5% C3&C5 removed human serum. The results shown in Figure 21 B indicate that no relevant reduction of cell viability occurred when complement activation was impaired, confirming the dependence of complement for antibody-induced cytotoxicity.

DISCUSSION

The results of this study indicated that the biparatopic antibody DuoHexabody-CD37 was able to achieve the best levels of CDC among the tested articles in both of the target cell lines analysed. In particular, it showed dramatic effects on Daudi cells, where cell viability was reduced by around 90% already at the concentration of 0,4 pg/mL.

This result is in line with previous observations which demonstrated that DuoHexaBody-37 had superior CDC activity compared to other CD37 antibodies. This bispecific antibody can deliver better CDC through its improved ability to induce IgG hexamerization via its E430G mutation and dual epitope targeting.

The data shown revealed that the antibodies tested activated CDC in a higher extent in Daudi than in Raji cells. One of the reasons behind this observation might be the higher expression of CD37 in Daudi cells (mention paper/report showing this), which would promote a larger number of antigenantibody interactions and make these cells more sensitive to the complement cascade. In addition, the results indicated that the NNV candidates worked differently in the two cell lines and suggested that different molecular mechanisms might be prominent for CDC in the two systems. The reasons underlying these differences are not known and might regard the charge and glycan and lipid composition of the plasma membrane.

In general, the NNV humanized antibodies displayed modest cytotoxicity when compared to DuoHexabody-CD37 and the positive control Ofatumumab. However, differences among the candidates could be appreciated and conclusions based on the Fc modifications characterizing the antibodies could be drawn. The NNV candidates that worked best in Raji cells are NNV024 and NNV032, two afucosylated antibodies. Despite their limited ability to deliver CDC in comparison to DuoHexabody-CD37, their performance is clearly superior to the one of NNV023, NNV025 and NNV030, which do not present any modification and did not induce any cytotoxicity. Looking at the CDC rates of the NNV antibodies bearing REW mutations (NNV029, NNV031 and NNV032), it seems that, in this system, Fc afucosylation is the prominent factor in the induction of CDC. As a matter of fact, the addition of REW mutations on NNV024, which gave rise to NNV032, did not improve, or actually slightly decreased its CDC performance. When comparing NNV031 and its afucosylated variant NNV032, a reduction of cell viability could be observed, suggesting that, in Raji cells, Fc afucosylation could be determinant for CDC. This observation is supported by the fact that the cytotoxicity levels induced by NNV024 and NNV032 were on the same range of another afucosylated antibody, the commercially approved Obinutuzumab.

The best NNV antibodies in Daudi cells were NNV029 and NNV031 , two candidates bearing REW mutations able to decrease cell viability levels by around 40% at the concentration of 10 pg/mL. As shown in example 2 the NNV antibodies with REW mutations showed an increased interaction with the complement protein C1q and could have a better potential in CDC activation. Therefore, the superior CDC ability exhibited by NNV029 and NNV031 compared to NNV023, NNV025 and NNV030 might be due to an improved interaction with C1q and a better complement fixation on Daudi cell surface. Although Fc afucosylation enhanced CDC induction (NNV024 and NNV032 vs NNV023, NNV025 and NNV030), it seemed to contrast the effect of REW mutations when both modifications were present. In fact, the ability of NNV032 to induce CDC was lower than the one shown by its fully fucosylated counterpart NNV031. The analysis of the CDC induction by Obinutuzumab clearly showed that both REW mutant and afucosylated NNV antibodies performed better. The data obtained with Obinutuzumab are consistent with published research showing very limited ability to promote complement activation {Hernandez, Nature, Obinutuzumab for the treatment of non-Hodgkin lymphomas} and data shown in example 2.

The analysis of CDC activation for antibodies where the C-terminal lysine was clipped did not show any relevant changes. The comparison of NNV030 (lysine clipped) with its unclipped version NNV023 did not reveal any differences in cell viability. Previous reports {van den Bremer et al., mAbs, 2015} suggested that this modification could contribute to maximal complement activation, but this was not observed in the current study.

CONCLUSION

The screening of NNV antibodies showed that, at least in the concentration range tested, the CDC potential of the anti-CD37 humanized antibodies of the present disclosure is lower than the one displayed by Duohexabody-CD37 in both Daudi and Raji cell lines. On the other hand, the comparison of the results indicated that the performance of the afucosylated candidates (NNV024 and NNV032) is similar to the one of Obinutuzumab in Raji cells and that NNV antibodies bearing REW mutations (NNV029 and NNV031) achieve higher CDC induction than Obinutuzumab in Daudi cells.

EXAMPLE 9 Plasma half-life and pharmacokinetics of humanized anti-CD37 antibody variants in human FcRn expressing mice

AIM

The aim of the experiments was to compare the plasma half-lives (T1/2) and derive the PK parameters for a panel of engineered humanized anti-CD37 IgG 1 variants (NNV) side-by-side with obinutuzumab (anti-CD20, lgG1 , afucosylated, Gazyvaro) and a recombinant form of DuoHexabody-CD37 (anti-CD37; Biparatopic lgG1 ; DuoHexabody-CD37) in a pre-clinical state-of- the-art human FcRn transgenic mouse model.

A major contributor to the efficacy of monoclonal IgG therapeutics is its naturally long plasma halflife, which varies from 6-32 days in humans. In addition, the half-life of monoclonal IgG can be extended beyond this by Fc-engineering for improved pH-dependent binding to human FcRn. Both the efficacy, plasma half-life and pharmacokinetics (PK) of engineered IgGs are evaluated in mouse models during pre-clinical development. However, due to large cross-species differences in binding between FcRn and IgG, wild-type (WT) mice are not reliable models for evaluation of in vivo plasma half-life and PK parameters of humanized Fc-engineered IgG molecules. For example, human IgG has higher affinity for mouse FcRn than the human form, resulting in longer circulatory persistence than that of mouse IgG. In contrast, mouse IgG does not bind human FcRn and, as such, is not rescued from intracellular degradation, and therefore is cleared rapidly in human FcRn transgenic mice. Nevertheless, human FcRn transgenic mice are the “golden standard” for evaluation of human IgG therapeutic candidates. These mice lack representative amounts of endogenous IgG, which in humans are about 10-12 mg/mL. Naturally, this represents a significant competitive pressure on the IgG binding site of FcRn that influences the plasma half-life and PK parameters of injected IgG therapeutic candidates. To account for this, human FcRn transgenic mice can be pre-loaded with intravenous immunoglobulin (Mg) to mimic competition. Under such conditions, differences in plasma half-life and PK parameters between IgG variants, which otherwise could be masked, can be measured.

MATERIALS AND METHODS

The study was performed as outlined in the protocol NNV020-p-076-2020.08.02. Each of the antibodies (see Table 21 ) was administered by intravenous (i.v) injection to hemizygous human FcRn transgenic mice (Tg32 hemizygous, the Jackson Laboratory (JAX)) (5 animals per group) at a dose of 5 mg/kg. In addition, NNV023 and NNV029 (lgG1-WT and lgG1-REW) were administered by i.v injection to Tg32 hemizygous mice (5 animals per group 15 mg/kg) pre-loaded with 250 mg/kg IVIg 2 days before administration of the antibodies. Blood samples were taken and plasma isolated at day 1 , 2, 3, 5, 7, 10, 12, 16, 19, 23 and 30 post administration of the antibodies. The plasma concentration of the antibodies was determined by ELISA before half-life and PK parameters were calculated using two different methods. The in vivo experiments were performed at JAX Services, and the plasma samples analyzed at the Oslo University Hospital (OUH).

Table 21. Test antibodies

In vivo plasma half-life:

1. Hemizygous human FcRn transgenic mice (B6.Cg-Fcgrt^tm1Dcr Tg(FCGRT)32Dcr/DcrJ) (Tg32 hemizygous) that are knockout for the mouse FcRn heavy chain and express the genomic transgene of the human FcRn heavy chain, under the control of the human FcRn promotor, were used in the plasma half-life/pharmacokinetic study.

2. On day 0, the mice were dosed with 5 mg/kg of the antibodies, diluted in 1x phosphate buffered saline (PBS), by i.v injection. Where stated, the mice were pre-loaded with 250 mg/kg IVIg (privigen) by i.v injection at day -2 of the study. An overview of the study groups is shown in Table 22.

3. Blood samples (25 pl) were drawn from the retro-orbital sinus on day 1 , 2, 3, 5, 7, 10, 12, 16, 19, 23 and 30 post administration of the antibodies. For the two study groups in IVIg pre-loaded mice, blood samples were isolated up until day 23.

4. The blood samples were immediately mixed with 1 pl 1% K3-EDTA to prevent coagulation and centrifuged at 17000 x g for 5 min at 4°C to isolate plasma.

5. Isolated plasma was diluted 1 :10 in glycerol/PBS solution (1 :1) and stored at -20°C until analysis. JAX recommends analyzing the plasma samples within 6 months of isolation. 6. The concentrations of the antibodies in plasma, as well as total IgG levels in mice pre- loaded with IVIg, were measured by ELISA, as described below.

Table 22 Overview of study groups:

Quantification of antibodies in plasma from Tg32 hemizygous mice and total IgG in IVIg pre-loaded Tg32 hemizygous mice:

1. 96-well EIA/RIA plates were coated with 100 pl of anti-human IgG (whole molecule) antibody from goat diluted to 1 pg/mL in PBS, and incubated overnight at 4°C.

2. The plates were washed four times with PBS containing 0.05% Tween20 (T) using a Hydrospeed™ plate washer.

3. The remaining surface area was blocked with PBS/T containing 4% skimmed milk powder (S) for 1 hour (h) at room temperature (RT).

4. The plates were washed four times with PBS/T using a Hydrospeed™ plate washer.

5. The plasma samples were diluted 1 :50, 1 :100, 1 :200, 1 :400, 1 :800 and 1 :1600 in PBS/T/S, added to the plates and incubated for 1 hour at RT. A 12-point standard curve (STD) (1000.0 - 0.488 ng/mL) of the antibody variant corresponding to the study group being analyzed was added to the same plate in duplicates.

An example of the ELISA plate setup used to analyze the plasma samples from one mouse is shown in Table 23.

6. Antibodies captured from plasma samples were detected by an alkaline phosphatase (AP)- conjugated polyclonal anti-human IgG (Fc specific) antibody from goat (diluted 1 :5000 in PBS/T/S).

7. The plates were washed four times with PBS/T using a Hydrospeed™ plate washer. 8. Visualization was performed by addition of p-nitrophenylphosphate (1 mg/mL in diethanolamine buffer).

9. The plates were developed for 20-30 minutes before the 405 nm absorption values were recorded using a TECAN Sunrise plate reader. Table 23. ELISA setup used to analyze plasma samples.

Establishing an ELISA assay for detection of NNV023 and NNV029 in plasma from IVIg pre-loaded

Tg32 hemizygous mice:

1. 96-well EIA/RIA plates were coated with 100 pl of the anti-idiotypic Fab AbD34091_Fab (obtained from NNV) diluted to 0.5 pg/mL in PBS and incubated overnight at 4°C. AbD34091_Fab binds to the CDR region of the NNV antibodies.

2. The plates were washed four times with PBS/T using a Hydrospeed™ plate washer.

3. The remaining surface area was blocked with PBS/T/S for 1 hour at RT.

5. Concentration series of NNV023 or NNV029 (1000.0 - 0.488 ng/mL) were prepared in PBS/T/S containing a fixed concentration of 250 pg/mL IVIg and added to the plates in duplicate. In addition, IVIg only (250 pg/mL or 125 pg/mL) diluted in PBS/T/S and PBS/T/S only were included as controls.

6. The plates were incubated for 1 hour at RT before washing four times with PBS/T/S using a Hydrospeed™ plate washer.

7. Antibodies captured on AbD34091_Fab were detected by adding an AP-conjugated polyclonal anti-human IgG (Fc specific) antibody from goat (diluted 1 :5000 in PBS/T/S).

8. Visualization was performed by addition of p-nitrophenylphosphate (1 mg/mL final concentration in diethanolamine buffer).

9. The plates were developed for 30-40 minutes before the 405 nm absorption values were recorded using a TECAN Sunrise plate reader.

Quantification of antibodies in plasma from IVIg pre-loaded mice:

1. 96-well EIA/RIA plates were coated with 100 pl AbD34091_Fab diluted to 0.5 pg/mL in PBS and incubated overnight at 4°C.

3. The remaining surface area was blocked with PBS/T/S for 1 hour at RT.

5. Plasma samples were diluted 1 :50, 1 :100, 1 :200, 1 :400, 1 :800 and 1 :1600 in PBS/T/S, added to the plates. In addition, a 12-point standard curve (STD) of the antibody variant corresponding to the study group being analyzed (1000 - 0.488 ng/mL) was added in duplicates. The plates were incubated for 1 hour at RT before washed four times with PBS/T using a Hydrospeed™ plate washer.

6. Antibodies captured from the plasma samples were detected by adding an AP-conjugated polyclonal anti-human IgG (Fc specific) antibody from goat (diluted 1 :5000 in PBS/T/S).

7. The plates were washed four times with PBS/T using a Hydrospeed™ plate washer.

8. Visualization was performed by adding p-nitrophenylphosphate (1 mg/mL final concentration in diethanolamine buffer).

9. The absorbance values were recorded at 405 nm using a TECAN Sunrise plate reader.

Calculation of plasma half-life and PK parameters:

1. The absorbance values obtained from the ELISA analysis were interpolated to the standard curves using Graphpad Prism. Values corresponding to the exponential part standard curve were used. The concentration of the antibody samples in plasma at each time point was then calculated by multiplying with sample dilution factor used (Microsoft Excel). The first concentration measured (day 1) was then normalized to 100% and remaining data points were plotted as percent antibody remaining in plasma. Data points from the p-elimination phase were then used to calculate the plasma half-life using the formula:

where ti/2 is the half-life, Ac is the amount of antibody remaining, and Ao is the amount of antibody at day 1 and t is the elapsed time.

2. The antibody concentrations in plasma determined by ELISA were then fitted to a noncompartmental (NCA) PK model using the gPKPDsim PK add-on for MatLAb. Briefly, the antibody concentrations were plotted in Microsoft Excel and loaded into gPKPDsim before both half-life and PK parameters were calculated using predefined formulas. A summary of the PK parameters determined is shown in Table 24.

Table 24. Pharmacokinetic parameter output in gPKPDsim

Statistical Analysis:

Statistical analysis was performed using repeated measurements two-way ANOVA with Geisser- Greenhouse correction in GraphPad Prism. Outlier detection was performed using Grubb's test in GraphPad Prism.

RESULTS

First, the percentages of antibodies remaining in plasma from the Tg32 hemizygous mice over time, in the absence of competing IgG, were plotted and their plasma half-life calculated. Of note, one mouse from each of the NNV025, NNV024 and NNV030 groups was excluded from the analysis due to significantly lower levels of antibody (data not shown). This is likely due to errors made during administration. The results showed that the amino acid substitution (V110D) introduced to reduce the risk of immunogenicity in humans, increased the plasma half-life of NNV023 (12.0 ± 2.4 days) by more than 4 days compared to NNV025 (7.4 ± 0.8 days) (Figure 22A, Table 25). Afucosylation of NNV023 led to a more than 3-days reduction in plasma half-life, NNV024 (8.8 ± 0.8 days) (Figure 22B, Table 25). While introduction of the REW Fc substitutions, which enhance pH dependent human FcRn binding, did not alter the plasma half-life of NNV029 (12.00 ± 3.3 days) compared to NNV023 (Figure 22C, Table 25), removal of the C-terminal heavy chain lysine gave rise to a shorter plasma half-life, NNV030 (9.7 ± 0.7 days) (Figure 22D, Table 25). However, combining removal of the C-terminal lysine with the REW Fc substitutions restored the plasma half-life of NNV031 to the level of NNV023 and NNV029 (11.95 ± 2.1 days) (Figure 22E, Table 25).

In stark contrast, combining the REW Fc substitutions with afucosylation considerably reduced the plasma half-life of NNV032 (3.3 ± 0.5 days) compared to NNV023 (Figure 22F, Table 25). Finally, the plasma half-lives of obinutuzumab (4.4 ± 1.3 days) and DuoHexabody-CD37 (4.1 ± 1.2 days) were somewhat longer than NNV032, but nevertheless more than 7 days shorter than NNV023 (Figure 22G, Table 25).

Table 25. Plasma half-life values derived from percentages of antibody remaining in plasma from Tg32 hemizygous mice over time in the absence of competition

Next, the plasma concentrations of the antibody variants were plotted (Figure 23) and fitted to a non-compartmental analysis (NCA) PK model. The results from this analysis are summarized in Table 26. The results revealed a similar hierarchy between the antibody variants as obtained in the percentage-based analysis above. Again, NNV023 showed a 4-day extended plasma half-life compared to NNV025 (16.3 ± 5.8 days vs 12.1 ± 4.7), while afucosylation and removal of the C- terminal lysine (NNV024 and NNV030) led to a modest reduction in plasma half-life (12.3 ± 6.2 days and 12.6 ± 5.2 days). Combining removal of the C-terminal lysine with the REW Fc substitutions (NNV031) resulted in a half-life comparable with that of NNV023 (15.8 ± 8.2 days). Table 26. NCA PK parameters of antibodies in Tg32 hemizygous mice in the absence of competition.

Introduction of the REW Fc substitutions led to a modest increase in plasma half-life and reduced clearance for NNV029 (22.1± 3.3 days), in contrast to the percentage-based analysis. The NCA analysis showed, again, that NNV032, obinutuzumab and DuoHexabody-CD37 were cleared from plasma significantly faster than the other antibody variants. The half-lives of the antibody variants reflected differences in the AUC, clearance, mean residency time (MRT) and volume of distribution at steady state (Vss), as determined by the NCA PK model. The effect of competition on plasma half-life:

To further investigate the effect of the REW Fc substitutions, NNV023 and NNV029 were administered to Tg32 hemizygous mice (5 mg/kg I 5 animals per group) pre-loaded with 250 mg/kg IVIg to introduce natural competitive pressure on human FcRn.

Establishment of a specific ELISA:

For the plasma analysis, an ELISA assay was established to specifically detect NNV023 and NNV029 in presence of a high concentration of IVIg. Briefly, the anti-idiotype Fab fragment AbD34091 was coated in wells (0.5 pg/mL), followed by addition of titration series of NNV023 and NNV029 (1000.0 - 0.488 ng/mL) in the presence of a constant amount of IVIg (250 pg/mL). In addition, IVIg only (250 pg/mL or 125 pg/mL) or PBS/T/S (blank) samples were included as controls. Captured antibodies were detected with an AP-conjugated anti human IgG (Fc-specific) antibody from goat. (Figure 24A). The results showed that the ELISA assay could be used to specifically capture NNV023 and NNV029 (Figure 24B). While addition of 250 pg/mL IVIG did produce a weak background signal, the absorbance values obtained from 125 pg/mL IVIg was almost identical to that of the blank sample.

To determine whether the ELISA assay could be used to analyze plasma from IVIg pre-loaded mice, the total concentration of human IgG in the plasma samples was measured using a two-way anti-human IgG Fc ELISA. The result showed that the total IgG concentration in the plasma samples (IVIg + NNV IgG) over time was not statistically different between the groups (NNV023 and NNV029) and lower than 125 pg/mL, with a maximum concentration of 114.5 pg/mL (Figure 25). Notably, one mouse in the NNV023 group showed 3-fold higher levels of total IgG and one mouse in the NNV029 group showed 10-fold lower levels of total IgG than the remaining animals (data not shown). This was likely caused by errors made during pre-loading. These two animals were excluded from further analysis since the differences in total IgG levels could affect the results in the ELISA readout, and likely result in underestimation of the plasma half-life of NNV023 and overestimation of the half-life of NNV029. Both the higher level of total IgG in one mouse in the NNV023 group and the lower level of total IgG in one mouse in the NNV029 group, were detected as significant outliers using the Grubb's test when performed on the plasma concentrations at day 1 of the study (Table 27).

Table 27. Grubb's test, outlier elimination.

Plasma half-life in the presence of competition:

The established ELISA assay was then used to analyze the NNV023 (n=4) and NNV029 (n=4) plasma samples from IVIg pre-loaded Tg32 hemizygous mice. First, the percentage of antibodies remaining in plasma over time was determined and the plasma half-lives calculated. The results showed an almost 2-fold extended plasma half-life for NNV029 (9.8 ± 3.1 days) compared to NNV023 (5.5 ± 1.3 days) (Figure 26A, Table 28). Notably, the half-lives were 2.2-fold and 1.2-fold shorter than in the absence of competition. Furthermore, and importantly, the plasma concentration of NNV029 was between 2.5 and 3.3-fold higher than that of NNV023 between day 1 and day 12 and 4.8-5.6-fold higher between day 16 and day 23 (Figure 26B). The concentrations of NNV023 and NNV029 in plasma were then fitted to the NCA PK model using gPKPDsim (Table 29), where the analysis revealed a more than 2-fold increase in plasma half-life for NNV029 (17.2 ± 8.2 days) compared to NNV023 (7.2 ± 5.1 days). NNV029 also showed higher AUC, Cmax and MRT as well as slower clearance than NNV023.

Table 28. Plasma Half-life values derived from percentages of antibody remaining in plasma from IVIg pre-loaded Tg32 hemizygous mice over time.

Table 29. NCA PK parameters ofNNV023 and NNV029 in IVIg pre-loaded Tg32 hemizygous mice.

DISCUSSION

The NNV023 antibody, engineered for reduced immunogenicity risk in humans, showed a long plasma half-life and favorable PK parameters in the Tg32 hemizygous mouse model in the absence of competing IqG (12.0 ± 2.4 days). Strikingly, this variant showed about 5 days longer plasma half-life than that of humanized NNV025 (7.4 ± 0.8 days), which is a result of only one amino acid substitution difference in the light chain (V110D). As such, this particular change alters the ability of the antibody to persist in the circulatory system. If this relates to altered engagement of human FcRn in vivo is not known. However, no difference in human FcRn binding and transport properties have been observed, as described in previous reports (also see comments below when tested in the presence of competing IgG).

By comparison, recombinant versions of the FDA approved antibodies bevacizumab (anti-VEGF; IgG 1 ; Avastin) and ustekinumab (anti-p40; IgG 1 ; Stelara) have demonstrated half-life values of 6.1 ± 0.6 days and 7.1 ± 1.9 days in the same mouse model when determined using the percentage method (Foss et al, manuscript in prep). Based on these data, NNV023 is efficiently rescued from intracellular degradation by human FcRn in vivo.

Both afucosylation (NNV024) and removal of the C-terminal lysine (NNV030) resulted in a modest reduction in plasma half-life (4 days) compared to NNV023. The reduced half-life of NNV024 is in line with a previous report showing a modest reduction in half-life of afucosylated trastuzumab (anti-HER2; lgG1 ; Herceptin). Furthermore, no increase in plasma half-life was observed in the context of the REW Fc substitutions (NNV029) in the absence of competition (12.0 ± 3.3 days), but the substitutions did restore the half-life of NNV031 (lacking the C-terminal lysine) (11.9 ± 2.1 days) to the level of NNV023. However, the plasma concentration of NNV029 was higher than that of NNV023 over time. In line with this, the NCA PK model gave rise to an almost 6 days longer plasma half-life for NNV029 (22.1± 3.3 days) compared to NNV023 (16.3 ± 5.8 days). Of note, as the NCA PK model is based on the plasma concentrations, the s.d values are higher than that derived from the percentage model where the amount of antibody in plasma is normalized to 100% on day 1.

Combination of afucosylation and the REW Fc substitutions (NNV032) resulted in significantly shorter plasma half-life (3.3 ± 0.5 days). The early and rapid decline in the plasma levels of NNV032, observed both in the percentage and concentration analysis, together with its poor NCA PK profile, suggests that stability in plasma or other off-target effects may be involved rather than its depletion by anti-drug antibodies (ADAs). The latter is usually measured as a sharp break in the linear clearance curve 7-12 days post administration. However, generation of ADAs may be measured by an anti-mouse IgG ELISA, or by performing the study in human FcRn transgenic SCID mice.

To mimic a more natural environment with competition from endogenous IgG, Tg32 hemizygous mice were pre-loaded with IVIg.

The results showed that the presence of competition dramatically altered the plasma half-lives and PK profiles of NNV023 and NNV029. Specifically, the REW Fc substitutions increased the plasma half-life of NNV029 by 1.8-fold in the percentage analysis and its plasma concentration was between 2.5 and 3.3-fold higher than that of NNV023 between day 1 and day 12. Towards the end of the experiment, between day 16 and day 23, the plasma concentration of NNV029 was 4.8-5.6- fold higher than that of NNV023.

Interestingly, the favorable extended plasma half-life of NNV023 compared with that of NNV025 in the absence of competition, 12.0 ± 2.4 days vs 7.4 ± 0.8 days, was drastically reduced to 5.5 ± 1.3 days in the presence of competing IgG. As such, the favorable effect of V110D on plasma half-life was not resistant to competition for the IgG binding site on FcRn. In stark contrast, the REW substitutions became dominant in the presence of IVIg.

Furthermore, using the NCA PK model the plasma half-life of NNV029 was 2.4-fold longer, its MRT 2.5-fold longer and its clearance rate 2.8-fold slower compared to NNV023. The results are comparable to unpublished data using the same experimental setup (Foss et al, manuscript in prep). Taken together, NNV029 is rescued more efficiently by human FcRn in vivo than NNV023 in the presence of natural competition. Of note, in the absence of competition, NNV031 , which contains the REW Fc substitutions and lacks the C-terminal lysine, showed a similar half-life as that of NNV029 (only with the REW substitutions).

CONCLUSION

The REW Fc substitutions were shown to prolong the plasma half-life of NNV029 in the presence of natural competition for human FcRn. In addition, the concentration of NNV029 was several folds higher in plasma over time. Therefore, NNV029 demonstrated the most favorable in vivo profile of the panel of antibodies tested, and should therefore be the lead candidate for in vivo therapy experiments.

Example 10

Therapeutic effect of NNV024, NNV029, NNV031, NNV032, duohexabdy-CD37 and obinutuzumab in Tg mice injected with intravenous Daudi cells

AIM

The aim of this study is to compare the therapeutic effect of a single injection of NNV024, NNV029, NNV031 and NNV032 with duohexabdy-CD37 and obinutuzumab in Tg mice injected with intravenous Daudi cells. The model chosen for this study is a transgenic strain of SCID mice expressing the human FcRn (SCID FcRn-/- hFcRn (32) Tg), which represent the gold standard for the characterization of human IgG therapeutic candidates. Through the pre-loading of normal human IgG, this model can mimic the competition of IgGs for FcRn that is determinant for plasma half-life. Therefore, using these mice as disease hosts would combine the evaluation of both the pharmacokinetic profile and the therapeutic efficacy of the diverse antibody candidates in vivo.

MATERIALS AND METHODS

Test articles: Vials containing the necessary amounts of the humanized monoclonal antibodies NNV024 (7,4 mg/ml), NNV029 (5 mg/ml), NNV031 (4 mg/ml), NNV032 (5 mg/ml) (Nordic Nanovector), Duohexabody-CD37 (9,35 mg/ml) (manufactured in the Nordic Nanovector laboratory), obinutuzumab antibody (Gazyvaro, Roche) (5 mg/ml diluted in 0.9% NaCI from 25 mg/ml stock), and 0.9% NaCI were provided. All solutions were sterile filtered. Normal human Immunoglobinfor intravenous injection (100 mg/mL) (IVIg, Privigen, CSL Behring) were used for pre-dosing of the mice.

Animals and housing: 58 female SCID FcRn-Z- hFcRn (32) Tg mice, 5-8 weeks old (5 weeks old = 5 mice, 6 weeks old = 13 mice and 8 weeks old = 40 mice), were ordered from The Jackson Laboratory (018441 - B6.Cg-Fcgrt<tm1Dcr> Prkdc<scid> Tg(FCGRT)32Dcr/DcrJ (jax.org)). The mice were allowed 5 days of acclimatization prior to study start. The mice were provided with filtered drinking water (Danmil filter, Product code DA25CSSS02 19252077, Rating 0.2 MIC http://danmil.dana5.dk/). Water bottles are changed 3 times a week. The mice were fed ad libidum with irradiated rodent diet (Altromin NIH#31 M -from Brogarden, Denmark). The animals were maintained with a 12 hours lighting cycle at a room temperature of 21 - 23°C and air relative humidity of 40%.

Cell culturing: Daudi cells were grown in T75 flasks (Thermo) with 25 ml medium/per flask, in 37°C, 5% CO2 humidified incubator. They will be harvested when 1-1.5 millions cells per ml, viability minimum 85% at harvest. Cell culture medium RPMI-1640 - GlutaMAX (Life Technologies Cat no 72400-021 ) supplemented with 10% inactivated FBS (Life Technologies cat no 10270), 1% PenStrep (Life Technologies cat no 15140-122) and 1 mM Sodium Pyruvate (Sigma cat no p5280).

Injection of cells: The mice were injected with tumor cells suspended in RPMI medium from a fresh bottle without any supplements. The mice were injected in the lateral tail vein with 10 million cells pr. animal in a volume of 100 pl per mouse. The animals were specifically observed for 15-20 minutes to register any treatment related adverse effects.

Experimental design: The current study includes 7 study groups, each with 8-9 animals per group. The treatment groups will be according to the Table 30. This study will run as a “blinded” study, so the study director and the pathologist will not know what treatment each group will be receiving.

Table 30. Study groups.

On day -1 the animals were weighed and randomized into 7 groups according to body weight and age so that each group had the same average body weight and similar distribution of age at study start. Subsequently, serum blood samples were drawn (100 pl whole blood) from all mice and all the animals were pre-ldosed with 250 mg/kg normal human IgG (IVIg, Privigen) to mimic competition with endogenous IgG on Fc receptors.

On day 0 the mice were inoculated intravenously with 10 million Daudi cells and the next day (day 1), treatment were initiated according to Table 1.

Animals were euthanized at one or more of the following humane endpoinds: Hind leg paralysis, overall weight loss of > 20% or weight loss of > 15% over a period of one week if signs of discomfort, kidney tumors 7 - 10 mm in diameter or larger, or signs of substantial discomfort. Prior to euthanasia serum blood samples were collected and the serum stored at -20°C. After the mice have been euthanised necropsy were performed.

RESULTS

One week after start of treatment all mice in all treatment groups were alive (Table 31).

Table 31. Surviving fraction 1 week after start of treatment.

One week after start of treatment the average body weight of the mice in the treatment groups were similar (Table 32).

Table 32. Average bodyweight of mice after start of treatment.

DISCUSSION & CONCLUSION

The results are still being evaluated. Long-term treatment results are presented in Example 13.

Example 11 - Comparison of ADCP induced by NNV023, NNV029, NNV031 and NNV032, DuoHexabody-CD37, rituximab and obinutuzumab in a panel of Non-Hodgkin lymphoma cell lines.

AIM

The goal of this study was to measure the ability of the anti-CD37 antibodies NNV023, NNV029, NNV031 and NNV032 to induce Antibody-dependent Cell Phagocytosis (ADCP) on a panel of CD37-expressing target cells from different subtypes of non-Hodgkin lymphoma (NHL). The candidates were tested on three Burkitt’s lymphoma cell lines (Daudi, Ramos and Raji), five diffuse large B-cell lymphoma (DLBCL) cell lines (DOHH-2, U2932, SU-DHL-4, SU-DHL-6 and WSU- DLCL-2) and two mantle cell lymphoma (MCL) cell lines (Granta-519 and REC-1). In addition, the Acute Lymphocytic Leukemia (ALL) cell line REH was included as a negative control due to its low CD37 expression. The ADCP potential of these antibodies was evaluated by using a bioluminescent reporter assay quantifying the activation of ADCP pathway activation on effector cells expressing either FcyRlla-R131 or H131 variants. This study also aimed to compare the ADCP potential of the humanized NNV antibodies against the approved anti-CD20 therapeutic antibodies obinutuzumab (Gazyvaro, Roche) and rituximab (MabThera, Roche), and against recombinant DuoHexabody-CD37 (HexaBody®-CD37 or GEN-3009, Genmab), a biparatopic anti- CD37 antibody currently in Phase l/ll of clinical testing.

MATERIALS AND METHODS

Table 33 presents an overview of the test items for which ADCP capacity was evaluated.

Table 33: Antibodies used in the study.

Culturing of target cells

The Daudi, Ramos, Raji, Rec-1 , and REH cell lines were obtained from American Type Culture Collection (ATCC, Manassas, VA). The U2932, SU-DHL-4, SU-DHL-6, WSU-DLCL-2 and Granta- 519 cell lines were provided by University Medical Center Groningen (UMCG, The Netherlands). DOHH-2 cells were purchased from German Collection of Microorganisms and Cell Cultures GmbH (DSMZ, Braunschweig, Germany). Authentication of cell lines was performed at Eurofins Genomics Europe Applied Genomics GmbH (Ebersberg, Germany). The genetic features of all tested lines were found to match their characteristics in the databases.

Daudi, Ramos, Raji, DOHH-2, U2932, SU-DHL-4, SU-DHL-6, WSU-DLCL-2 and REC-1 cell lines were cultured in RPMI1640 (Gibco, Waltham, USA) supplemented with GlutamaxX (Gibco, Paisley, UK), 10% heat-inactivated Fetal Bovine Serum (Gibco) and 1% Penicillin-streptomycin mix (Gibco). Granta-519 cells were grown in high-glucose DMEM supplemented with GlutamaxX (Gibco, Paisley, UK), 10% heat-inactivated FCS (Gibco) and 1 % Penicillin-streptomycin mix (Gibco). Cells were incubated in a humidified atmosphere with 5% CO2 at 37°C. The cell suspensions were usually diluted to a concentration of 0,5 million cells/mL with pre-warmed medium twice a week. Three days before the start of the ADCP experiment, all the cell lines were diluted to ensure that they were in the exponential phase of growth at the beginning of the experiment.

Experiment procedure in short

On the day of the experiment, the target cells (T) (Daudi, Ramos, Raji, Rec-1 , U2932, DOHH-2, SU-DHL-4, SU-DHL-6, WSU-DLCL-2 and Granta-519) were harvested, washed three times in PBS with 0.5% BSA, and resuspended in RPMI supplemented with 4% low IgG serum (assay buffer) to the concentration of 1.0 x 10⁶ cells/mL. From this dilution, 25 pL were seeded (2.5x10⁴ cells/well) in each well of a white flat bottom 96 well plate (Thermo Fisher). Right after cell seeding, the plates were conditioned to room temperature (21 °C, RT) for 15 min. Serial dilutions (1 :10 dilutions covering the range 0.001 - 1000 ng/mL in assay buffer for the ADCP assay with R131 variant and 1 :3 dilutions covering the interval 1-10000 ng/mL for H131 variant) of the test antibodies NNV023, NNV029, NNV031 , NNV032, obinutuzumab, rituximab, and DuoHexabody-CD37 were added to the wells as duplicates and incubated for 30 min at 21 °C to induce opsonization. Some wells containing target cells were left without antibodies (untreated control cells) and served for the estimation of background signal. The Promega effector Jurkat cells (E) stably expressing either FcyRlla-R131 or H131 receptors were thawed, resuspended in the assay buffer and added to the target cells in proportion 3:1 (E:T). The system was incubated for 16-18 hours at 37 °C 5% CO2 before the Bio-Gio Luciferase Assay reagent (Promega) was added to the plate in equivolumetric ratio with the content of the wells. After 15 min of incubation at RT, the luminescence read-out was performed using a Spark microplate reader (TECAN, Switzerland). The values in relative luminescent units (RLU) were obtained for each well containing cells.

Data analysis

For each target cell line, the value of the background signal (the mean RLU obtained from triplicates of untreated control cells) was withdrawn from the signals of all wells of the same plate. The mean RLU and standard deviation of duplicates for each test antibody concentration were calculated. Normalized induction of ADCC was calculated by dividing the luminescence values of the treated cells by the mean luminescence of untreated (Ab-free) cells. The data obtained for each antibody concentration were fitted to a sigmoidal 4PL curve using GraphPad 9.3. Each data point and their standard deviations (SD) together with the resulting interpolation spline lines were plotted in graphs representing the Iog10 [Ab] (pg/mL) on the x axis and the relative ADCP activation on the y axis.

Calculation of the area under the curve (AUC) for ADCP induction was performed using GraphPad Prism 9.3. The results for each cell line were represented in the corresponding clustered scatter plot containing the antibody name on the x axis and AUC on the y axis. The average value for these parameters was calculated for each Ab and over imposed on the clustered scatter plot.

RESULTS

For this study, the humanized anti-CD37 candidates, obinutuzumab, rituximab and DuoHexabody- CD37 were tested in a panel of ten NHL cell lines. The lymphoma models used as target cells are representative of different B-NHL subtypes, such as Burkitt’s lymphoma (Ramos, Raji and Daudi), GCB- (DOHH-2, SU-DHL-4, SU-DHL-4 and WSU-DLCL-2) and ABC-DLBCL (U2932), and MCL (Rec-1 and Granta-519). The degree of induction of ADCP signalling in vitro measured through the reporter assay (NF-AT associated luciferase activation) was dependent on the target cell line.

The results obtained upon treatment of each cell line included in the panel, using ADCP effector cells stably expressing the allotypes R131 and H131 of FcyRlla are described in the next two sections.

ADCP induction in presence of effector cells expressing FcyRlla-R131

The results of a single experiment investigating the ADCP activation using the Promega ADCP assay with FcyRlla-R131 variant are presented in Figure 29, 30, 31 and 32.

Among the NNV antibodies, the REW-containing candidates NNV029, NNV031 and NNV032 showed the best ADCP activation in presence of FcyRlla-R131 expressing effector cells (Figure 29, 30, 31 and 32). In particular, in presence of this isoform of FcyRlla, NNV032 achieves the best ADCP induction levels among the NNV antibodies. When comparing the performance of the wild- type anti-CD37 humanized antibody NNV023 and of the REW containing IgGs, it is possible to appreciate that the AUC of NNV029, NNV031 and NNV032 is around two-fold the one of NNV023 (mean AUC NNV023 = 5,34 vs NNV029 = 10,13; NNV031 = 10,02; NNV032 = 11 ,78).

As shown in Figure 32, the average AUC values for the REW-containing antibodies are on a similar range as the one of obinutuzumab (mean AUC ratio: NNV029/obinutuzumab = 0,86; NNV031/obinutuzumab = 0,85; NNV032/obinutuzumab = 0,996), especially for the afucosylated antibody NNV032. This observation suggests an overall comparable ADCP performance. However, a closer look to the graphs in Figure 29, 30 and 31 and to the scatter plots in Figure 32 shows some variation among the tested cell lines. In fact, the humanized anti-CD37 candidates are inducing higher levels of ADCP than Obinutuzumab in the Burkitt’s lymphoma cell line Daudi (Figure 29). However, the AUC values of the REW containing candidates and obinutuzumab are similar in the other two Burkitt’s lymphoma models (Raji and Ramos), in the DLBCL cell lines DOHH-2, U2932 and WSU-DLCL-2 and in the MCL model Rec-1. The performance of obinutuzumab is clearly superior when ADCP induction is assessed in the MCL cell line Granta- 519 and in the DLBCL models SU-DHL-4 and SU-DHL-6.

The two benchmarking antibodies rituximab and DuoHexabody-CD37 display very modest activation of ADCP in all the NHL cell lines of the panel. As observed in Figure 29, 30, 31 and 32, their performances are similar to the one shown by NNV023 (mean AUC ratio: rituximab/NNV023 = 1 ,11 ; DuoHexabody-CD37/NNV023 = 1 ,09) and clearly lower than those of obinutuzumab (mean AUC ratio: rituximab/obinutuzumab = 0,5; DuoHexabody-CD37/DuoHexabody-CD37 = 0,49) and the REW-containing antibodies (mean AUC rituximab = 5,95; DuoHexabody-CD37 = 5,82 vs NNV029 = 10,13; NNV031 = 10,02; NNV032 = 11 ,78).

ADCP induction in presence of effector cells expressing FcyRlla-H131

The results of a single experiment investigating the ADCP activation using the Promega ADCP assay with FcyRlla-H131 variant are presented in Figure 33, 34, 35 and 36.

As observed in the previous section, the REW-containing antibodies NNV029, NNV031 and NNV032 were able to induce ADCP with a higher extent than the wild-type candidate NNV023 (Figure 33, 34, 35 and 36). Overall, their activation capacity, as described by the AUC values, was 6-7 fold higher than the one displayed by NNV023 (mean AUC ratio: NNV029/NNV023 = 7,2; NNV031/NNV023 = 7,2; NNV032/NNV023 = 6,3). In contrast with the results from the previous section, in presence of FcyRlla-H131 variant, the fully fucosylated candidates NNV029 and NNV031 show a slightly superior performance when compared to the afucosylated candidate NNV032. The comparison of the mean AUC values (Figure 36) for all the tested items shows that NNV029 and NNV031 are overall the best antibodies in the panel (mean AUC NNV029 = 50,9; NNV031 = 51 ). In presence of FcyRlla-H131 expressing effector cells, obinutuzumab, rituximab and DuoHexabody-CD37 displayed comparable levels of ADCP activation (mean AUC rituximab = 42,8; obinutuzumab = 43; DuoHexabody-CD37 =42,1 ). Moreover, the ADCP performance of these antibodies is similar to the one displayed by NNV032 (AUC NNV032 = 44,61). As reported in the previous section, variability among the cell lines was observed. Remarkably, obinutuzumab is the best performing antibody in Granta-519 and SU-DHL-4, rituximab and DuoHexabody-CD37 display the highest AUC in U2932 and WSU-DLCL-2, while NNV029 and NNV031 show the best ADCP in Daudi, Ramos, Rec-1 and SU-DHL-6.

DISCUSSION

The analysis of ADCP induction in a panel of NHL cell lines performed in a reporter system using effector cells expressing the receptors either FcyRlla-131H or 131 R variants suggests that REW mutations are a key factor in enhancing ADCP activation. Comparing the ADCP performance achieved by the REW mutated antibodies NNV029, NNV031 and NNV032 and their wild-type counterpart NNV023, these modifications in the Fc part are crucial to improve the interaction with FcyRlla and activate its downstream signaling. This observation is in line with the interaction studies reported in Example 2.

In presence of FcyRlla-131R expressing effector cells, NNV032 was the best antibody in the panel, displaying ADCP activation levels similar to the ones of obinutuzumab and performing clearly better than rituximab and DuoHexabody-CD37. In contrast, when the effector cells expressed the H131 allotype, NNV029 and NNV031 showed the best ADCP induction, while NNV032, obinutuzumab, rituximab and DuoHexabody-CD37 achieved similar performances. The differences observed in the ADCP effects in presence of the two FcyRlla-131 allotypes suggests that there might be differences in the interaction with the Fc of the various antibodies, which might influence the activation of downstream ADCP pathways.

Variations in ADCP activation have also been observed among the different target cell lines. A possible explanation for this fact could be due to differences in the expression of CD37 and CD20. For instance, the high ADCP activation reported for REW-containing NNV antibodies in Daudi cells could be due to the high CD37 expression levels shown by this cell line [Dahle, J., et al., Evaluating antigen targeting and anti-tumor activity of a new anti-CD37 radioimmunoconjugate against non-Hodgkin's lymphoma. Anticancer Res, 2013. 33(1): p. 85-95.]. However, Fc modifications and the interaction with FcyRlla are crucial as well, as shown by the lower effects induced by NNV023 and DuoHexabody-CD37. CONCLUSION

In presence of FcyRlla-131R expressing effector cells, NNV032 was the best antibody in the panel, displaying ADCP activation levels similar to the ones of obinutuzumab and performing clearly better than rituximab and DuoHexabody-CD37.

When the ADCP effector cells expressed the H131 allotype of FcyRlla, NNV029 and NNV031 showed the best ADCP induction, while NNV032, obinutuzumab, rituximab and DuoHexabody- CD37 achieved similar performances.

Example 12 - Comparison ofADCC induced by NNV023, NNV029, NNV031 and NNV032, DuoHexabody-CD37, rituximab and obinutuzumab in a panel of Non-Hodgkin’s lymphoma cell lines

AIM

The goal of this study was to measure the ability of the anti-CD37 antibodies NNV023, NNV029, NNV031 and NNV032 to induce Antibody-dependent Cellular Cytotoxicity (ADCC) on a panel of CD37-expressing target cells from different subtypes of non-Hodgkin lymphoma (NHL). The candidates were tested on three Burkitt’s lymphoma cell lines (Daudi, Ramos and Raji), five diffuse large B-cell lymphoma (DLBCL) cell lines (DOHH-2, U2932, SU-DHL-4, SU-DHL-6 and WSU- DLCL-2) and two mantle cell lymphoma (MCL) cell lines (Granta-519 and REC-1). In addition, the Acute Lymphocytic Leukemia (ALL) cell line REH was included as a negative control due to its low CD37 expression. The ADCC potential of these antibodies was evaluated by using a bioluminescent reporter assay quantifying the activation of ADCC pathway activation on effector cells expressing FcyRllla-V158 variant. This study also aimed to compare the ADCC potential of the humanized NNV antibodies against the approved anti-CD20 therapeutic antibodies obinutuzumab (Gazyvaro, Roche) and rituximab (MabThera, Roche), and against recombinant Duohexabody-CD37 (HexaBody®-CD37 or GEN-3009, Genmab), a biparatopic anti-CD37 antibody currently in Phase l/ll of clinical testing.

MATERIALS AND METHODS

Test antibodies

Table 34 presents an overview of the test items for which ADCC capacity was evaluated.

Table 34: Antibodies used in the study.

Culturing of target cells

Daudi, Ramos, Raji, DOHH-2, U2932, SU-DHL-4, SU-DHL-6 and WSU-DLCL-2, REC-1 and REH cell lines were cultured in RPMI1640 (Gibco, Waltham, USA) supplemented with GlutamaxX (Gibco, Paisley, UK), 10% heat-inactivated Fetal Bovine Serum (Gibco) and 1% Penicillinstreptomycin mix (Gibco). Granta-519 cells were grown in high-glucose DMEM supplemented with GlutamaxX (Gibco, Paisley, UK), 10% heat-inactivated FCS (Gibco) and 1% Penicillin-streptomycin mix (Gibco). Cells were incubated in a humidified atmosphere with 5% CO2 at 37°C. The cell suspensions were usually diluted to a concentration of 0,5 million cells/mL with pre-warmed medium twice a week. Three days before the start of the ADCC experiment, all the cell lines were diluted to ensure that they were in the exponential phase of growth at the beginning of the experiment.

Experimental procedure

On the day of the experiment, the target cells (T) (Daudi, Ramos, Raji, Rec-1 , U2932, DOHH-2, SU-DHL-4, SU-DHL-6, WSU-DLCL-2, Granta-519, and REH) were harvested, washed three times in PBS with 0.5% BSA, and resuspended in RPMI supplemented with 4% low IgG serum (assay buffer) to the concentration of 1.0 x 10⁶ cells/mL. From this dilution, 25 pL were seeded (2.5x10⁴ cells/wel I) in each well of a white flat bottom 96 well plate (Thermo Fisher). Right after cell seeding, the plates were conditioned to room temperature (21°C, RT) for 15 min. Serial dilutions (0.001 - 1 pg/mL in assay buffer) of the test antibodies NNV023, NNV029, NNV031 , NNV032, obinutuzumab, rituximab, and DuoHexabody-CD37 were added to the wells as duplicates and incubated for 30 min at 21 °C to induce opsonization. Some wells containing target cells were left without antibodies (untreated control cells) and served for the estimation of background signal. The Promega effector Jurkat cells (E) stably expressing FcyRllla-V158 receptors were thawed, resuspended in the assay buffer and added to the target cells in proportion 3:1 (E:T). The system was incubated for 16-18 hours at 37 °C 5% CO2 before the Bio-Gio Luciferase Assay reagent (Promega) was added to the plate in equivolumetric ratio with the content of the wells. After 15 min of incubation at RT, the luminescence read-out was performed using a Spark microplate reader (TECAN, Switzerland). The values in relative luminescent units (RLU) were obtained for each well containing cells.

Data analysis

For each target cell line, the value of the background signal (the mean RLU obtained from triplicates of untreated control cells) was withdrawn from the signals of all wells of the same plate. The mean RLU and standard deviation of duplicates for each test antibody concentration were calculated. Normalized induction of ADCC was calculated by dividing the luminescence values of the treated cells by the mean luminescence of untreated (Ab-free) cells. The data obtained for each antibody concentration were fitted to a sigmoidal 4PL curve using GraphPad 9.3. Each data point and their standard deviations (SD) together with the resulting interpolation spline lines were plotted in graphs representing the Iog10 [Ab] (pg/mL) on the x axis and the relative ADCC activation on the y axis.

Within the sigmoidal 4PL regression model fitting, the potency (EC50) and efficacy (Emax) to induce ADCC signaling were calculated. Moreover, calculation of the area under the curve (AUC) was performed using GraphPad Prism 9.3. The results for each cell line were represented in the corresponding clustered scatter plot containing the antibody name on the x axis and relevant parameter (EC50, Emax, AUC) on the y axis. The average value for these parameters was calculated for each Ab and over imposed on the clustered scatter plot.

RESULTS

The results of a single experiment investigating the ADCC activation induced by the test antibodies in a panel of eleven cell lines are presented in Figure 37, 38, 39 and 40. The lymphoma lines used as target cells are models of B-NHL subtypes with an unmet therapeutical need, such as Burkitt’s lymphoma (Ramos, Raji and Daudi), GCB- (DOHH-2, SU-DHL-4, SU-DHL-4 and WSU-DLCL-2) and ABC-DLBCL (U2932), and MCL (Rec-1 and Granta-519). An acute lymphoblastic leukemia (ALL) cell line, REH, was used as a CD20- ZCD37- control [Schneider, D., et al., A tandem CD19/CD20 CAR lentiviral vector drives on-target and off-target antigen modulation in leukemia cell lines. J Immunother Cancer, 2017. 5: p. 42.]. The degree of induction of ADCC signaling in vitro was dependent on the target cell line. Among the NNV candidates, NNV032 displayed overall the best ADCC activation (Emax, EC50 and AUC) in all the cell lines in the panel (Figures 37, 38, 39 and 40). When comparing the parameters describing the ADCC concentration-response of NNV032 with the other NNV antibodies, these show around 77% of the efficacy shown by NNV032 (mean Emax ratio: NNV023/NNV032 = 0,77; NNV029/NNV032 = 0,77; NNV031/NNV032 = 0,80), around 4 times lower potency (mean EC50 ratio: NNV023/NNV032 = 4,45; NNV029/NNV032 = 4,47; NNV031/NNV032 = 5,1 1 ) and 63% of the AUC (mean AUC ratio: NNV023/NNV032 = 0,64; NNV029/NNV032 = 0,62; NNV031/NNV032 = 0,63), suggesting that NNV023, NNV029 and NNV031 act as partial agonists in ADCC induction. Being fully afucosylated, NNV032 and obinutuzumab showed a high degree of agonism to FcyRllla-158V in all the tested cell lines. The ability to activate ADCC measured in this reporter assay is overall similar, with comparable relative ADCC activation curves (Figure 37, 38 and 39), mean Emax (Emax NNV032 = 30,17 vs obinutuzumab = 30,95) and AUC (AUC NNV032 = 103,3 vs obinutuzumab = 102,9) (Figure 40A and 40C). However, NNV032 displays better potency than obinutuzumab in all the cell lines except Rec-1 (Figure 40B), with an average EC50 1.52-fold lower. Rituximab and DuoHexabody-CD37 displayed a low ADCC potential compared to NNV032 and obinutuzumab. As represented in Figure 40, they showed the highest mean EC50 values (27,02 ng/mL for rituximab and 11 ,89 ng/mL for DuoHexabody-CD37) and the lowest mean AUC (43,74 for rituximab and 40,52 for DuoHexabody-CD37) among the tested antibodies. The efficacy of rituximab was comparable to the values shown by NNV023, NNV029 and NNV031 (mean Emax rituximab = 23,46 vs NNV023 = 23,35, NNV029 = 23,28, NNV031 = 24,44), while DuoHexabody- CD37 presented the lowest efficacy (mean Emax = 17,65).

DISCUSSION

N-glycan antibody afucosylation is a key factor in enhancing ADCC induction. Afucosylation of NNV031 to obtain NNV032 was able to improve ADCC activation by the tested humanized anti- CD37 candidate. This modification was crucial to achieve ADCC induction levels similar to the ones shown by the afucosylated anti-CD20 antibody obinutuzumab and maximize the complementarity of Fc parts of NNV032 and FcyRllla receptors.

Since different technologies for antibody afucosylation are currently available, antibodies may be manufactured with a different level of afucosylation and different composition of N-glycan within their Fc part. Obinutuzumab and NNV032 were manufactured using different afucosylation technologies by different manufacturers and, although their ADCC performance is similar, NNV032 has a slightly higher potency. This observation might be explained by qualitative and quantitative differences in fucose content at the Fc part of each antibody.

While low fucose content improved the ADCC performance of anti-CD37 humanized antibodies, the presence of REW mutations did not change the agonistic properties of the NNV antibodies, as observed when comparing the NNV023 with the REW-mutant candidates NNV029 and NNV031. In the same manner, clipping of the C-terminal lysine did not influence the ADCC activation by NNV031.

CONCLUSION

The afucosylated humanized anti-CD37 antibody NNV032 was found superior to the other NNV antibodies, rituximab and DuoHexabody-CD37 in inducing ADCC signaling upon treatment of NHL cell lines. Moreover, NNV032 showed similar efficacy and AUC, but better efficacy compared to obinutuzumab.

Example 13 - In vivo comparison of the therapeutic efficacy ofNNV029, NNV031 and NNV032 with Duohexabody-CD37 and obinutuzumab in a Daudi xenograft lymphoma model

AIM

The aim of the present study is to compare the humanized CD37-targeting antibodies NNV029, NNV031 and NNV032 against obinutuzumab and recombinant Duohexabody-CD37 in their ability to treat mice injected with Daudi tumor cells. The model selected as host of Daudi cell xenograft is a transgenic strain of SCID mice expressing the human FcRn (SCID FcRn-Z- hFcRn (32) Tg), which represent the gold standard for the characterization of human IgG therapeutic candidates. Through pre-loading with normal human IgG, the competition of IgGs for FcRn determining plasma half-life can be mimicked. Therefore, the use of these mice as disease hosts allows to combine the evaluation of both the pharmacokinetic profile and the therapeutic efficacy of the diverse antibody candidates in vivo.

MATERIALS AND METHODS

Test articles

Table 35: Test articles used in the study.

Vials containing the necessary amounts of each test article in Table 35 were provided to the ArcticLas facility, in Reykjavik, Iceland, where the animal study was performed. Moreover, normal human Immunoglobin for intravenous injection (100 mg/mL) (IVIg, Privigen, CSL Behring) for pre- loading and formulation buffer (control solution, 0.9% NaCI) were used. All the solutions were sterile filtered and stored at 2-8 C until preparation of the injectates.

Cell culturing

The lymphoma cell line (Daudi) was cultured at Keldur, Institute for experimental Pathology, University of Iceland. Suspension cells were grown in T75 flasks (Thermo), 25 ml medium/per flask, at 37°C, in a 5% CO2 humidified incubator. They were harvested when 1-1.5 millions cells per ml, viability minimum 85% at harvest. Cell culture medium RPMI-1640 - GlutaMAX (Life Technologies) was supplemented with 10% inactivated FBS (Life Technologies) and 1% PenStrep (Life Technologies).

Animals

50 female SCID FcRn-Z- hFcRn (32) Tg mice, 5-8 weeks old, were obtained from The Jackson Laboratory (018441 - B6.Cg-Fcgrt<tm1Dcr> Prkdc<scid> Tg(FCGRT)32Dcr/DcrJ (jax.org)). The mice were acclimated for two weeks prior to study start, housed in mice IVC-rack (Green line Sealsafe cages from Tecniplast, Italy) five mice per cage and kept on Alpha Dry irradiated paper bedding (from Brogarden, Denmark). The mice were provided with filtered drinking water (Danmil filter) and fed ad libitum with irradiated rodent diet (Altromin NIH#31 M -from Brogarden, Denmark). The animals were maintained with a 12-hour lighting cycle at a room temperature of 21 - 23°C and air relative humidity of = 40%.

Procedure in short

One day prior to inoculation (day -1 ), the animals were weighed and randomized into six study groups (see Table 2) according to body weight and age so that each group would have the same average body weight and similar distribution of age at study start. On this day, serum blood samples were drawn (100 pl whole blood) from all mice and all the animals were pre-loaded with normal human IgG. The mice were then treated intravenously with 250 mg/kg normal human IgG (IVIg, Privigen) to mimic competition with endogenous IgG on Fc receptors.

On day 0 the mice were inoculated in the lateral tail vein with 10 million Daudi cells resuspended in 100 pL of RPMI without supplements. The animals were specifically observed for 15-20 minutes to register any treatment related adverse effects. The next day (day 1 ), intravenous injection of the test articles was performed according to Table 36.

One day after inoculation the mice received 6 different treatments (8-9 mice per group, see table 36). All treatment groups received a dose of 2.69 mg/kg. A humane end point was reached when one or more of the following clinical findings were present: Hind leg paralysis, Weight loss of > 20% or other signs of substantial discomfort.

Table 36: Study groups. Pre-dosing, treatment regimen, therapy dose, number of mice per group and dosing frequencies.

After inoculation, Daudi cells distributed to lymph nodes, kidneys, lungs, ovaries, bone, bone marrow and spine and meninges, eyes, muscle and brown adipose tissue. To describe the progression of the disease, the mice were weighed weekly the first two weeks after inoculation and at least twice a week after that.

The animals were inspected daily, and clinical signs of disease registered. Animals were euthanized at one or more of the following humane endpoinds: hind leg paralysis, overall weight loss of > 20% or weight loss of > 15% over a period of one week if signs of discomfort, kidney tumors 7 - 10 mm in diameter or larger, or signs of substantial discomfort. Prior to euthanasia serum blood samples were collected and the serum stored at -20°C. After the mice have been euthanized, necropsy and collection of organs for histopathology analysis were performed.

The experiment was scheduled to continue until a statistical difference was found between corresponding groups of the different drugs or until it was clear that no statistical difference would be attained by prolonging the experiment. 130 days after cell inoculation, the study was terminated and a necropsy and histopathology analysis of all the remaining mice were conducted to look for tumors and tumor infiltration in the organs affected by the disease.

RESULTS

The data were analyzed for survival (humane endpoint was hind leg paralysis or weight loss of > 20%) using Log-rank (Mantel-Cox Test (GraphPad Prism 8.3.0)), listed in Table 37 and represented in the Kaplan Meier survival plot of Figure 27. The statistical pairwise comparison of all test groups was performed using Holm-Sidak method (a= 0,05).

Figure 27 shows the survival of the mice treated with the different antibodies as a function of time after injection of the treatments. The first animals in the control group were euthanized 55 days after inoculation and the last animal with clinical signs died after 95 days, leaving one animal symptom-free until study termination.

The overall survival analysis (Table 37 showed that all treatment groups had statistically significant better survival than the control group (0.9% NaCI), p = 0.0004 (Groups NNV029, NNV032, Duohexabody-CD37 and obinutuzumab) and p=0.0023 (NNV031) (Kaplan Meier: Log-rank (Mantel-Cox Test)). There was not a statistically significant better survival between any of the treatment groups (p>0.05) (Kaplan Meier: Log-rank (Mantel-Cox Test)).

Table 37: Median survival of 6 groups of female SCID FcRn-/- hFcRn (32) Tg mice with i.v. injected Daudi-lymphoma cells (10 million cells).

*One animal in NNV031 treatment group died spontaneously during the study with a mediastinal tumor suspected to be a spontaneous thymic lymphoma not associated with Daudi cells.

The column “% Animals without Daudi cell lymphoma at day 130” in Table 37 indicates for each group the percentage of animals surviving until study termination (day 130) which, after necropsy and histopathology analysis, were deemed Daudi lymphoma-free.

Figure 28 presents mean body weights of each treatment group from arrival (day -14) until the study ended (Day 130). Overall, the body weight plots follow the trend of the survival curves of each treatment group. All groups had a similar weight gain pattern for the first four and a half weeks, but from day 35, the mean body weight of the control group started decreasing as the animals started showing signs of disease. The body weight curves for the remaining treatment groups were similar with small fluctuations up until day 111 , when treatment group NNV029 started losing weight. As two animals started showing signs of disease, the groups mean body weight started to decline.

DISCUSSION

The aim of the present study was to investigate the efficacy potential of the humanized CD37- targeting antibodies NNV029, NNV031 and NNV032 and compare them to the well-established CD20-targeting antibody obinutuzumab and to the recombinant CD37-targeting biparatopic antibody Duohexabody-CD37. Based on data from ADCC and ADCP in vitro experiments (reported in examples 3 and 4) and on the pharmacokinetic properties shown in example 9, the goal was to evaluate whether the NNV antibodies have a better therapeutic efficacy than obinutuzumab and recombinant DuohexabodyCD37 in an animal model mimicking disseminated B-cell malignancy. The results show that a single treatment, according to body weight (2.69 mg/kg), with the humanized CD37-targeting antibodies NNV029, NNV031 and NNV032 appears to be equally as effective in treating Daudi-induced disease as obinutuzumab and Duohexabody-CD37 (Genmab). There was not a statistically significant better survival between any of the treatment groups (p>0.05). All treatment groups had a statistically significant better survival than the Control group, p = 0.0004 (all groups but NNV031) and p=0.0023 (NNV031) (Kaplan Meier: Log-rank (Mantel-Cox Test)).

CONCLUSION

The results show that a single injection, according to body weight (2.69 mg/kg), of the humanized CD37-targeting antibodies NNV029, NNV031 or NNV032 appears to have similar therapeutic efficacy in treating disseminated B-cell malignancy as obinutuzumab and recombinant DuoHexabody-CD37.

Claims

1. An antibody, antibody fragment or antibody derivative thereof, which comprises, a) a heavy chain variable domain (VH) comprising VH-CDR1 , VH-CDR2 and VH-CDR3, and b) a light chain variable domain (VL) comprising VL-CDR1 , VL-CDR2 and VL-CDR3, wherein, c) the heavy chain variable domain (VH) comprises the amino acid sequence of any one of SEQ ID NOs: 1 [heavy chain of H02871], wherein, according to SEQ ID NO: 1 , position 2, or position 11 is I or V, position 12 is V or K, position 38 is K or R, position 48 is M or I, position 68 is A or V, position 70 is I or L, position 72 is R or V, position 81 is I or M, and wherein i. the heavy chain VH-CDR1 comprises the amino acid sequence GYSFTD, ii. the heavy chain VH-CDR2 comprises the amino acid sequence PYN, iii. the heavy chain VH-CDR3 comprises the amino acid sequence PYGHYAM, d) the light chain variable domain (VL) comprises the amino acid sequence of any one of SEQ ID NOs: 8 [light chain of H02871], wherein, according to SEQ ID NO: 8, position 13 is A or T, position 43 is A or S, position 49 is Y or N, position 71 is F or Y, position 78 is M or L, position 106 is I, M or V, position 110 is V or D, i. the light chain VL-CDR1 comprises the amino acid sequence ASQDVST, ii. the light chain VL-CDR2 comprises the amino acid sequence WA, iii. the light chain VL-CDR3 comprises the amino acid sequence HYSTP, e) a lambda or kappa light chain constant domain of human origin, and f) an IgG 1 , lgG2, lgG3 or lgG4, IgM, IgA, IgE or IgD heavy chain constant domain of human origin, wherein the heavy chain constant domain comprises one or more amino acid sequences variants(s), at one or more positions selected from the group consisting of 311 , 434, 428, 438, and 435, with reference to the IgG 1 heavy chain constant domain of SEQ ID NOs: 21 [heavy chain constant domain of lgG1], or synonymous positions in lgG2, lgG3, lgG4, IgM, IgA, IgE or IgD.

2. The antibody, antibody fragment or antibody derivative thereof according to claim 1 , wherein the antibody, antibody fragment or antibody derivative thereof is an anti-CD37 antibody, antibody fragment or antibody derivative thereof.

3. The antibody, antibody fragment or antibody derivative thereof according to any of claims 1 or 2, wherein the antibody is a monoclonal antibody. The antibody, antibody fragment or antibody derivative thereof according to any one of the preceding claims, wherein the antibody fragment is a fragment selected from the group consisting of an scFV and scFv-Fc fragment. The antibody, antibody fragment or antibody derivative thereof according to any one of the preceding claims, wherein the heavy chain variable domain (VH) comprises the amino acid sequence of any one of SEQ ID NOs: 1-7 [VH sequence of AH02871 , AH02875, AH02877, AH02879, AH02886 and AH02895] and the light chain variable domain (VL) comprises the amino acid sequence of any one of SEQ ID NOs: 8-18 [VL sequences of AH02871 , AH02875, AH02877, AH02879, AH02886, AH02895, AH02877J106M, AH02877J106V,

AH02877 V110D, AHO2877_I1O6M_V110D and AH02877J106V V110D], The antibody, antibody fragment or antibody derivative thereof according to any one of the preceding claims, wherein the heavy chain variable domain (VH) comprises the amino acid of SEQ ID NO: 2 [VH sequence of AH02871] and the light chain variable domain (VL) comprises the amino acid sequence of any one of SEQ ID NO: 10, 14-18 [VL sequences of AH02877, AH02877J106M, AH02877J 106V, AH02877_V110D, AHO2877_I1O6M_V110D and AH02877J106V V110D]. The antibody, antibody fragment or antibody derivative thereof according to any one of the preceding claims, wherein the heavy chain variable domain (VH) comprises the amino acid of SEQ ID NO: 2 [VH sequence of AH02871] and the light chain variable domain (VL) comprises the amino acid sequence of SEQ ID NO: 16 [VL sequences of AH02877 V110D]. The antibody, antibody fragment or antibody derivative thereof according to any one of the preceding claims, wherein the predicted immunogenicity risk score (IRS) of the VH domain according to any one of SEQ ID NOs: 1-7 is lower than the predicted IRS of SEQ ID NO: 19 [VH of Lilotomab]. The antibody, antibody fragment or antibody derivative thereof according to any one of the preceding claims, wherein the predicted immunogenicity risk score (IRS) of the VL domain according to any one of SEQ ID NOs: 8-18 is lower than the predicted IRS of SEQ ID NO: 20 [VL of Lilotomab]. The antibody, antibody fragment or antibody derivative thereof according to any of the preceding claims, wherein said amino acid sequences variants(s), is/are selected from the group consisting of lgG1-Q311R/N434W/M428E, lgG1-Q311R/N434W, lgG1-Q311R, lgG1- N434W, lgG3(b)-Q311R/N434W/M428E, lgG3(b)-Q311R/N434W/Q438E/R435H, lgG1- M252S/Q31 1 R/N434W/M428E, lgG1-Q311 R/N434P/M428E, lgG1-Q311 R/N434W/M428D, lgG1-Q311 R/N434W/M428E/H433K, lgG1-L309K/Q311 R/N434W/M428E, lgG1- L309R/Q311 R/N434W/M428E, lgG1-L309S/Q311 R/N434W7M428E, and lgG3(b)- Q311 R/N434W/M428E/R435H.

11 . The antibody, antibody fragment or antibody derivative thereof according to any one of the preceding claims, wherein the antibody comprises a kappa light chain constant domain of human origin and a IgG 1 heavy chain constant domain of human origin, and optionally wherein the IgG 1 heavy chain constant domain further comprises the Q194R/N317W/M311 E amino acid sequences variants(s), with reference to SEQ ID NO: 21.

12. The antibody, antibody fragment or antibody derivative thereof according to any one of the preceding claims, wherein the antibody comprises a kappa light chain constant domain of human origin and a IgG 1 heavy chain constant domain of human origin, and optionally wherein the IgG 1 heavy chain constant domain further comprises the Q311 R/N434W/M428E amino acid sequence variants, according to SEQ ID NO: 22.

13. The antibody, antibody fragment or antibody derivative thereof according to any one of the preceding claims, wherein the heavy chain constant region of said immunoglobulin has an amino acid sequence selected from the group consisting of SEQ ID NOs: 22-34.

14. The antibody, antibody fragment or antibody derivative thereof according to any one of claims 13-17, wherein said amino acid sequences variants(s), extends the serum half-life of the immunoglobulin as compared to an immunoglobulin lacking said mutation.

15. The antibody, antibody fragment or antibody derivative thereof according to any one of the preceding claims, wherein the heavy chain constant domain has an amino acid sequence according to any one of SEQ ID NOs: 22-34, and wherein the C-terminal residue is K or absent.

16. The antibody, antibody fragment or antibody derivative thereof according to any one of the preceding claims, wherein the C-terminal Lysine in the heavy chain constant domain according to any one of SEQ ID NOs 21-34, is absent and/or removed.

17. The antibody, antibody fragment or antibody derivative thereof according to any one of the preceding claims, wherein removal of the C-terminal K in the heavy chain constant domain according to any one of SEQ ID NOs 21-34, increases the antibody receptor binding of said antibody, wherein the antibody receptor is one or more receptors selected from the group consisting of FcRn, FcyRI, FcyRlla, FcyRllaH131 , FcyRllaR131 , FcyRllb, FcyRllla, FcyRlllaV158, FcyRlllaF158 and FcyRlllb, compared to an antibody, antibody fragment or antibody derivative thereof comprising said C-terminal Lysine. The antibody, antibody fragment or antibody derivative thereof according to any one of the preceding claims, wherein the amino acid sequence of said antibody, antibody fragment or antibody derivative thereof is a combination of heavy chain and light chain fragments, where said antibody, antibody fragment or antibody derivative comprises, a) a light chain having an amino acid sequence which is SEQ ID NO: 37 and a heavy chain having an amino acid sequence which is SEQ ID NO: 43 [NNV029 heavy chain, AH2871+REW mutations], b) a light chain having an amino acid sequence which is SEQ ID NO: 37 and a heavy chain having an amino acid sequence which is SEQ ID NO: 44 [NNV030 heavy chain, AH2871+ delCT-Lys], or c) a light chain having an amino acid sequence which is SEQ ID NO: 37 and a heavy chain having an amino acid sequence which is SEQ ID NO: 45 [NNV031 heavy chain, AH2871+REW mutations+delCT-Lys]. The antibody, antibody fragment or antibody derivative thereof according to any one of the preceding claims, wherein the antibody, antibody fragment or antibody derivative thereof is glycosylated. The antibody, antibody fragment or antibody derivative thereof according to claim 23, wherein the glycosylation of said antibody, antibody fragment or antibody derivative thereof is fucose deficient. The antibody, antibody fragment or antibody derivative thereof according to any one of claims 23-24, wherein the fucose deficient antibody, antibody fragment or antibody derivative thereof have an enhanced and/or improved induction of antibody-dependent cell-mediated cytotoxicity (ADCC), compared to a non-fucose deficient antibody, antibody fragment or antibody derivative thereof. The antibody, antibody fragment or antibody derivative thereof according to any one of the preceding claims, wherein the antibody, antibody fragment or antibody derivative thereof is a human or humanized antibody. The antibody, antibody fragment or antibody derivative thereof according to claim 22, wherein said human or humanized antibody have an enhanced and/or improved induction of antibodydependent cell-mediated cytotoxicity (ADCC), antibody dependent cellular phagocytosis (ADCP) and/or complement-dependent cytotoxicity (CDC) compared to a non-humanized antibody comprising the light chain of SEQ ID NO: 35 [NNV003] and the heavy chain of SEQ ID NO: 36 and/or the therapeutic antibody selected from the group consisting of Rituximab, Obinutuzumab and duohexabody-CD37, optionally in Daudi and/or Ramos cells.

24. The antibody, antibody fragment or antibody derivative thereof according any one of claims 22- 24, said human or humanized antibody have an enhanced and/or improved induction of antibody-dependent cell-mediated cytotoxicity (ADCC), antibody dependent cellular phagocytosis (ADCP) and/or complement-dependent cytotoxicity (CDC) and/or extends the serum half-life compared to a non-humanized antibody comprising the light chain of SEQ ID NO: 35 [NNV003] and the heavy chain of SEQ ID NO: 36 and/or the therapeutic antibody Rituximab optionally in Daudi and/or Ramos cells.

25. The antibody, antibody fragment or antibody derivative thereof according to any one of the preceding claims, wherein the antibody has an affinity for human CD37 expressing cells below 10 nM, such as below 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM and/or such as below 1 nM, such as below 900 pM, 800 pM, 700 pM, 600 pM, 500 pM, 400 pM or 331 pM.

26. A nucleic acid sequence encoding an antibody, antibody fragment or antibody derivative thereof according to any one of the preceding claims.

27. The nucleic acid sequence encoding an antibody, antibody fragment or antibody derivative thereof according to claim 26.

28. The nucleic acid sequence encoding a variable light chain and/or variable heavy chain of an antibody according to any one of claims 1-25.

29. A nucleic acid construct comprising one or more nucleic acid sequence(s) according to any one of claims 26-28.

30. A host cell comprising one or more nucleic acid sequence(s) according to any one of claims 26-28 and/or nucleic acid construct(s) according to claim 29.

31 . The host cell according to claim 30, wherein the host cell is a mammalian cell selected from the group consisting of Chinese hamster ovary (CHO) cells, CHO-K1 , CHO-DG44, mouse myeloma (NS0) cells, baby hamster kidney (BHK) cells, and human embryonic kidney lines (HEK293) cells, or an insect cell.

32. The host cell according to any one of claims 30-31 , capable of producing an antibody, antibody fragment or antibody derivative thereof according to any one of claims 1-25, wherein the cellular fucose glycosylation pathway of said host cell is modulated, such that the host cell produces a fucose deficient antibody, antibody fragment or antibody derivative thereof.

33. An antibody, antibody fragment or antibody derivative thereof according to any according to any one of claims 1-25, produced in a host cell according to any one of claims 30-31 .

34. An antibody, antibody fragment or antibody derivative thereof, drug conjugate that binds to human CD37 comprising: d) an antibody, antibody fragment or antibody derivative thereof according to any one of claims 1-25, e) a linker, and f) a drug selected from the group consisting of a toxin, a radioisotope, an anticancer drug, a cytotoxic drug and a cytostatic drug.

35. An antibody, antibody fragment or antibody derivative thereof, drug conjugate according to claim 34 wherein the linker is a chelating linker.

36. An antibody, antibody fragment or antibody derivative thereof, drug conjugate according to claim 34-35 wherein the linker is a chelating linker selected from the group consisting of p- SCN-bn-DOTA, DOTA-NHS-ester and p-SCN-Bn-TCMC.

37. The antibody, antibody fragment or antibody derivative thereof drug conjugate according to any of claims 35-36, wherein drug is a radionuclide, selected from the group consisting of ²¹¹At, 2¹³Bi, ²¹²Bi, ²¹²Pb, ²²⁵Ac, ²²⁷Th, ¹⁶¹Tb ⁹⁰Y, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁹⁹Au, ¹⁹⁴lr, ¹⁶⁶Ho, ¹⁵⁹Gd, ¹⁵³Sm, ¹⁴⁹Pm, 1⁴²Pr, ¹¹¹Ag, ¹⁰⁹Pd, ⁷⁷As, ⁶⁴Cu, ⁶⁷Cu, ⁴⁷Sc, and ¹⁷⁷Lu.

38. The antibody, antibody fragment or antibody derivative thereof, drug conjugate according to any of claims 34-37, wherein drug is an anticancer drug.

39. A pharmaceutical composition comprising, as the active ingredient, one or more antibody/antibodies, antibody fragment(s) or antibody derivative(s) thereof according to any one of claims 1-25 and/or an antibody, antibody fragment or antibody derivative thereof drug conjugate according to any one of claims 34-37, and a pharmaceutically acceptable carrier.

40. The pharmaceutical composition according to claim 39, wherein the composition further comprises an additional therapeutic agent, preferably selected in the group consisting of alkylating agents, angiogenesis inhibitors, antibodies, antimetabolites, antimitotics, antiproliferatives, antivirals, aurora kinase inhibitors, apoptosis promoters (for example, Bcl-2 family inhibitors), activators of death receptor pathway, Bcr-Abl kinase inhibitors, BiTE (Bi- Specific ? cell Engager) antibodies, antibody drug conjugates, biologic response modifiers, Bruton's tyrosine kinase (BTK) inhibitors, cyclin-dependent kinase inhibitors, cell cycle inhibitors, cyclooxygenase-2 inhibitors, DVDs, leukemia viral oncogene homolog (ErbB2) receptor inhibitors, growth factor inhibitors, heat shock protein (HSP)-90 inhibitors, histone deacetylase (HDAC) inhibitors, hormonal therapies, immunologicals, inhibitors of inhibitors of apoptosis proteins (lAPs), intercalating antibiotics, kinase inhibitors, kinesin inhibitors, Jak2 inhibitors, mammalian target of rapamycin inhibitors, microRNAs, mitogen-activated extracellular signal- regulated kinase inhibitors, multivalent binding proteins, non-steroidal antiinflammatory drugs (NSAIDs), poly ADP (adenosine diphosphate)-ribose polymerase (PARP) inhibitors, platinum chemotherapeutics, polo-like kinase (Plk) inhibitors, phosphoinositide-3 kinase (PI3K) inhibitors, proteasome inhibitors, purine analogs, pyrimidine analogs, receptor tyrosine kinase inhibitors, retinoids/deltoids plant alkaloids, small inhibitory ribonucleic acids (siRNAs), topoisomerase inhibitors, ubiquitin ligase inhibitors, hypomethylating agents, checkpoints inhibitors, peptide vaccine and the like, epitopes or neoepitopes from tumor antigens, as well as combinations of one or more of these agents.

41 . A method for producing an antibody, antibody fragment or antibody derivative thereof according to any one of claims 1-25, the method comprising, e) introducing into a mammalian host cell one or more nucleic acid construct(s) of claim 29, f) culturing said host cell in a suitable media, g) recovering said antibody, antibody fragment or antibody derivative thereof from the culturing broth, and h) purifying the antibody, antibody fragment or antibody derivative thereof.

42. The method according to claim 41 , wherein the host cell is a mammalian cell selected from the group consisting of Chinese hamster ovary (CHO) cells, CHO-K1 , CHO-DG44, mouse myeloma (NSO) cells, baby hamster kidney (BHK) cells, and human embryonic kidney lines (HEK293) cells, or an insect cell.

43. The method according to any one of claims 41-42, wherein the cellular fucose glycosylation pathway of said host cell is modulated, such that the host cell produces a fucose deficient antibody, antibody fragment or antibody derivative thereof.

44. A method of depleting CD37 expressing B-cells from a population of cells, comprising administering to said population of cells, an antibody, antibody fragment or antibody derivative thereof according to any one claims 1-25, an antibody, antibody fragment or antibody derivative thereof, drug conjugate according to claim 34-37, or a pharmaceutical composition according to any one of claims 39-40. 45. A method of treating disease, wherein targeting of CD37 expressing B-cells can provide an inhibition and/or amelioration of said disease, comprising administering a therapeutically effective amount of an antibody, antibody fragment or antibody derivative thereof according to any one claims 1-25, an antibody, antibody fragment or antibody derivative thereof, drug conjugate according to claim 34-37, or a pharmaceutical composition according to any one of claims 39-40.

46. A method of treating cancer and/or inflammatory disease(s) and/or autoimmune disease(s) comprising administering a therapeutically effective amount of an antibody, antibody fragment or antibody derivative thereof according to any one claims 1-25, an antibody, antibody fragment or antibody derivative thereof, drug conjugate according to claim 34-37, or a pharmaceutical composition according to any one of claims 39-40.

47. A method of treating cancer comprising administering a therapeutically effective amount of an antibody, antibody fragment or antibody derivative thereof according to any one claims 1-25, an antibody, antibody fragment or antibody derivative thereof, drug conjugate according to claim 34-37, or a pharmaceutical composition according to any one of claims 39-40.

48. A use of an antibody, antibody fragment or antibody derivative thereof according to any one claims 1-29, an antibody, antibody fragment or antibody derivative thereof, drug conjugate according to claim 38-42, or a pharmaceutical composition according to any one of claims 39- 40, in inhibiting cancer and/or inflammatory disease(s) and/or autoimmune diseases.

49. A use of an antibody, antibody fragment or antibody derivative thereof according to any one claims 1-25, an antibody, antibody fragment or antibody derivative thereof, drug conjugate according to claim 34-37, or a pharmaceutical composition according to any one of claims 39- 40, in ameliorating cancer and/or inflammatory disease(s) and/or autoimmune diseases.

50. An antibody, antibody fragment or antibody derivative thereof according to any one claims 1- 25, an antibody, antibody fragment or antibody derivative thereof, drug conjugate according to claim 34-37, or a pharmaceutical composition according to any one of claims 39-40, for use as a medicament.

51 . Use according to claim 50, wherein the medicament is for use in the treatment of cancer.

52. Use according to any one of claim 50-51 , wherein the medicament is for use in the treatment of B-cell malignancies. 53. Use according to any one of claims 50-52, wherein the medicament is for treating of a B-cell malignancy selected from the group consisting of B-cell non-Hodgkin’s lymphoma, B-cell chronic lymphocytic leukemia, hairy cell leukemia, lymphoplasmacytic lymphoma and multiple myeloma, comprising administering to the individual in need thereof, an effective amount of an antibody, antibody fragment or antibody derivative thereof according to any one claims 1-25, an antibody, antibody fragment or antibody derivative thereof, drug conjugate according to claim 34-37, or a pharmaceutical composition according to any one of claims 39-40.

54. Use according to claim 53, wherein the medicament is for treating of inflammatory and autoimmune diseases wherein CD37-positive B cells are enriched.

55. Use according to any of claim 50-54, wherein said medicament is administered once or sequential.

56. A formulation of an antibody, antibody fragment or antibody derivative thereof according to any one claims 1-25, an antibody fragment or antibody derivative thereof, drug conjugate according to claim 34-37, or a pharmaceutical composition according to any one of claims 39-40, for use in pre-treatment, wherein human CD37 is blocked in normal tissues before treatment with immunotoxic anti-CD37 or immunotoxic antibody-drug conjugate.

57. The formulation according to claim 56, wherein the formulation is suitable for administration by one or more administration routes selected from the group consisting of oral, topical, intravenous, intramuscular, and subcutaneous administration.

58. The formulation according to claim 57, wherein the amount of the antibody fragment or antibody derivative thereof according to any one claims 1-25, or the antibody fragment or antibody derivative thereof, drug conjugate according to claim 34-37, is at least 0.1 mg and not more than 1 g.

59. A kit for the production of an antibody fragment or antibody derivative thereof, drug conjugate according to any one of claims 34-37 comprising, c) two or more vials, wherein one vial contains a conjugate comprising a drug linked to a linker, and one vial comprising an antibody fragment or antibody derivative thereof according to any one claims 1-25, and d) optionally instructions for preparing said antibody-drug conjugate.

60. A kit for the production of an antibody fragment or antibody derivative thereof, drug conjugate according to any one of claims 34-37 comprising, c) two or more vials, wherein one vial contains a conjugate comprising a chelator linked to an antibody fragment or antibody derivative thereof according to any one claims 1- 25, a second vial containing a radionuclide, and d) optionally, instructions for preparing said antibody-radionuclide conjugate.

61 . An antibody, antibody fragment or antibody derivative thereof, conjugate that binds to human CD37 comprising: d) an antibody, antibody fragment or antibody derivative thereof according to any one of claims 1-25, e) a linker, and f) an compound enriched in one or more isotopes selected from the group consisting of 1¹C, ¹³N, ¹⁵O, ¹⁸F, ⁶⁴Cu and ⁸⁹Zr.

62. An antibody, antibody fragment or antibody derivative thereof, conjugate according to claim 61 , for use in positron emission tomography imaging.

63. The use according to claim 62, wherein the imaging is for providing diagnosis, staging, and monitoring treatment of cancers.

64. The use according to claim 63, wherein the cancer is B-cell non-Hodgkin’s lymphoma, B-cell chronic lymphocytic leukemia, hairy cell leukemia, lymphoplasmacytic lymphoma and multiple myeloma

65. A pharmaceutical composition, comprising an antibody fragment or antibody derivative thereof according to any one claims 1-25, or an antibody fragment or antibody derivative thereof, drug conjugate according to claim 34-37, further comprising one or more further molecule(s), wherein the further molecules is selected from the group consisting of one or more antibodies, small molecule(s), peptide(s) and toxin(s).