ANTIBODIES AND ANTIBODY CONSTRUCTS AGAINST CD38 CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to European Patent Application No.24173646.1, filed 1 May 2024, European Patent Application No. 24173650.3, filed 1 May 2024, GB Patent Application No. GB 2417959.0, filed 6 December 2024, and GB Patent Application No. GB 2417958.2, filed 6 December 2024, each of which is entirely incorporated herein by reference for all purposes. SUMMARY The present invention relates to antibodies against CD38 and uses thereof. In particular, the present invention relates to antibodies and antibody constructs that are directed against CD38 and to compositions, and in particular to pharmaceutical compositions, that comprise such antibodies and antibody constructs. The antibodies and antibody constructs against CD38 that are provided by the invention are also referred to herein as “antibodies of the invention”. The invention also relates to uses of such antibodies, antibody constructs and compositions in the prevention and treatment of diseases and disorders in human beings, and in particular in the prevention and treatment of diseases and disorders in human beings that can be prevented or treated by suitably administering, to a subject in need thereof, an antibody, antibody construct or composition as described herein (i.e. in one or more suitable amounts and according to a suitable dose regimen). Such diseases and disorders will be clear to the skilled person based on the disclosures herein and will also be referred to herein as “CD38- related diseases and disorders”. For example and without limitation, such CD38-related diseases and disorders may in particular include: (i) diseases and disorders that can be prevented and/or treated by suitably administering, to a subject in need thereof, one or more suitable amounts (i.e. according to a suitable dose regimen) of one of the known antibodies against CD38 referred to herein; and/or (ii) diseases and disorders that can be prevented and/or treated by suitably increasing, in the body of the subject to be treated, the activation of complement towards a cell that expresses CD38 on its surface (and in particular such that this may lead to an increase in the complement-dependent cytotoxicity that is exerted by the body of the subject to be treated towards a CD38 expressing cell). As further mentioned herein, such diseases and disorders may in particular include multiple myeloma (MM) and other forms of
cancer as well as other diseases or disorders for which known antibodies against CD38 are being applied or developed. The invention further relates to nucleic acids encoding the antibodies of the invention, (also referred to herein as “nucleic acids of the invention” or “nucleotide sequences of the invention”); to methods for preparing the antibodies of the invention; and to host cells expressing or capable of expressing the antibodies of the invention. Other aspects, embodiments, advantages and applications of the invention will become clear from the further description herein. The international application WO2019/238674 describes single domain antibodies for complement regulation which are capable of specifically binding to an epitope of a human complement factor selected from the group consisting of C1q, C3, C4 and/or the proteolytic derivatives C3b and C4b. WO2019/238674 also describes bi- and multispecific constructs comprising such a single domain antibody and at least one other antigen binding region (such as a second single domain antibody), in which said second antigen-binding domain binds to another target (such as a marker that is differentially expressed in cancer cells compared to non-malignant cells, a pathogenic marker a tissue-specific marker, an organ-specific marker, such as a marker specific for lung, eye, brain or kidney). WO2019/238674 also describes the use of such constructs in the treatment of diseases and disorders (depending, inter alia, on the target(s) of the antigen-binding regions that are present in said constructs next to the complement-binding single domain antibody or antibodies). The international application WO2019/238674 also describes a number of specific Nanobodies against C1q, including the Nanobodies called “IF75” (SEQ ID NO: 13 in WO2019/238674) and “IF78” (SEQ ID NO:17 in WO2019/238674). [Note: IF75 is also referred to herein as Nb75 and IF78 is also referred to herein as Nb78]. WO2019/238674 also describes that the Nanobodies described in this application may be humanized. The international application WO2020/167919 describes bispecific antigen-binding molecules comprising an antigen-binding domain that binds to a target antigen and an antigen- binding domain that binds to a complement component (such as C1q, C1r, C1s, C2, C3, C4, C5, C6, C7, C8 or C9). WO2020/167919 also describes the use of such bispecific antigen- binding molecules in the treatment of diseases and disorders (again depending, inter alia, on the target to which the target-binding antigen-binding domain in the molecule can bind). The non-prepublished International application entitled “Engineered complement engaging polypeptides” in the name of applicant and filed on the same date as the present application describes improved C1q binders that comprise, compared to a wild-type C1q
binder, one or more modifications resulting in reduced binding affinity to a C1q complement factor as compared to a binding affinity of said corresponding wild-type C1q binder. Some preferred C1q binders according to this International application may have a binding affinity to the C1q complement factor of from about 10 nanoMolar (nM) to about 2 microMolar (μM), as determined by biolayer interferometry, and/or may bind to the C1q complement factor with a KD from about 10 nM to about 1.5 μM, from about 50 nM to about 1.4 μM, from about 100 nM to about 1.3 μM, from about 150 nM to about 1.2 μM or from about 200 nM to about 1 μM. This International application also describes constructs comprising such C1q binders and at least one other antigen-binding moiety that binds to a target antigen, as well as uses of such constructs in the treatment of diseases and disorders (depending, inter alia, on the target antigen(s) to which said other antigen-binding moiety/moieties can bind). Said C1q binders may also be humanized. In particular aspects, said International application describes that such C1q binders can be generated by suitably introducing one or more alanine mutations into the CDRs of a naturally occurring single domain antibody (or into the CDRs of a suitable humanized variant of a naturally occurring single domain antibody). In some specific aspects, said International application describes such C1q binders that are variants of Nb75 or Nb78 (or of humanized variants of Nb75 or Nb78). The non-prepublished International application entitled “Engineered proteins that engage complement factor and a protein antigen, methods, and uses thereof” in the name of applicant and filed on the same date as the present application describes polypeptide constructs comprising: (a) a first complement factor-engaging domain that binds to C1q; (b) an antigen- binding moiety that binds to a target protein; and (c) a second complement factor-engaging domain, wherein the first complement factor-engaging domain and the second complement factor-engaging domain are linked to the antigen-binding moiety. In one particular embodiment, said International application describes antibodies or antibody-based constructs that comprise two antibody heavy chains and two antibody light chains, which antibodies comprise a first C1q binder that is linked or fused to one or the antibody heavy or light chains and second C1q binder that is linked or fused to another of the antibody heavy or light chains. As further described in more detail this International application, in such antibodies or antibody-based constructs, either the first C1q binder is linked or fused to one of the antibody heavy chains and the second C1q binder is linked or fused to the other antibody heavy chain or alternatively the first C1q binder is linked or fused to one of the antibody light chains and the second C1q binder is linked or fused to the other antibody light chain. As also described in more detail in said International application, in such antibodies or antibody-based constructs,
the C1q binders may be linked to the N-terminus or the C-terminus of the antibody heavy chain or light chain, or may be linked to the N-terminus or the C-terminus of the antibody heavy chain or light chain (and, according to a specific but non-limiting aspect, when they are linked to the heavy chain, preferably linked to the N-terminus of the heavy chain; and when they are linked to the light chain, preferably linked to the C-terminus of the light chain). As further described in this International application, according to one particular aspect, such antibodies or antibody-based constructs comprise two antibody heavy chains and two antibody light chains and two C1q binders, in which one of said C1q binders is linked or fused to the C- terminus of one or the antibody light chains and the second C1q binder is linked or fused to the C-terminus of the other antibody light chain. Pedersen et al., J Immunol (2023) 211 (3): 403–413, describe that “Fc-independent recruitment of C1 by modular bispecific single-domain Abs that simultaneously bind C1q and a surface Ag [which the authors of this reference also refer to as “BiCE”] can potently activate the complement system”, resulting in “superior ability of the bispecific Abs to induce complement-dependent cytotoxicity” which provides “effective complement activation and cell killing” and “a modality for potent complement activation”. Terms that are not specifically defined herein have the meaning given in these International applications (which are also referred to herein as the “co-pending International applications”). Provided herein are polypeptide constructs, wherein the polypeptide constructs comprise: at least one heavy chain and at least one light chain, wherein the at least one heavy chain and the at least one light chain bind to a cluster of differentiation 38 (CD38); and at least one complement factor-engaging domain that binds to a C1q complement factor, wherein an affinity of the at least one complement factor-engaging domain for the C1q complement factor is between 10 nanoMolar (nM) to about 2 microMolar (μM) as determined by biolayer inferometry. Further provided herein are polypeptide constructs, wherein the polypeptide construct comprises two complement factor-engaging domains that bind to the C1q complement factor. Further provided herein are polypeptide constructs, wherein the at least one complement factor-engaging domain comprise an affinity for the C1q complement factor that is about 0.1 μM up to about 2 μM as determined by biolayer inferometry. Further provided herein are polypeptide constructs, wherein the polypeptide construct comprises two heavy chains that bind to the CD38. Further provided herein are polypeptide constructs, wherein the polypeptide construct comprises two light chains that bind to the CD38. Further provided herein are polypeptide constructs, wherein the at least one heavy chain comprises an amino
acid sequence of SEQ ID NO: 1 or a variant thereof, wherein the variant comprises at least one amino acid substitution relative to SEQ ID NO: 1. Further provided herein are polypeptide constructs, wherein the at least one light chain comprises an amino acid sequence of SEQ ID NO: 2 or a variant thereof, wherein the variant comprises at least one amino acid substitution relative to SEQ ID NO: 2. Further provided herein are polypeptide constructs, wherein the at least one heavy chain comprises an amino acid sequence of SEQ ID NO: 1; and the at least one light chain comprises an amino acid sequence of SEQ ID NO: 2. Further provided herein are polypeptide constructs, wherein the two heavy chains each comprise two heavy chain variable regions (VH). Further provided herein are polypeptide constructs, wherein the two light chains each comprise two light chain variable regions (VL). Provided herein are polypeptide constructs, wherein the polypeptide constructs comprise a first heavy chain comprising SEQ ID NO: 1; a second heavy chain comprising SEQ ID NO: 1; a first light chain comprising SEQ ID NO: 2; a second light chain comprising: SEQ ID NO: 2; a first complement factor-engaging domain; and a second complement factor- engaging domain. Provided herein are pharmaceutical compositions, wherein the pharmaceutical composition comprises a polypeptide construct provided herein. Provided herein are methods of using an antibody or antibody construct or of a pharmaceutical composition provided herein in the prevention and/or treatment of a disease or disorder that can be prevented to treated by depleting, and/or suitably reducing the levels of and/or suitably controlling the level of, at least one cell or cell type expressing CD38 in the body of a subject in need of such prevention or treatment. Provided herein are methods of using an antibody or antibody construct or of a pharmaceutical composition provided herein in the prevention and/or treatment of a disease and disorder that can be prevented and/or treated by (suitably) increasing the complement- dependent cytotoxicity that is exerted by the body of the subject to be treated towards a CD38 expressing cell. BRIEF DESCRIPTION OF THE DRAWINGS The novel features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth
illustrative embodiments, in which the principles of the disclosed constructs are utilized, and the accompanying drawings of which: FIG.1 shows a schematic of Dara-BiCE, HexaBody-CD38, daratumumab, and a BiCE negative control antibody. FIG. 2 shows a graph of the cell lysis of various cell lines treated with the Dara-IgG- BiCE. The y-axis depicts percent maximum lysis, and the x-axis depicts cell lines grouped by activity as indicated. FIG.3 shows a graph of the cytotoxicity of WSU-DLCL2 cells treated with Dara-BiCE, HexaBody-CD38, daratumumab, and a BiCE negative control antibody. The y-axis depicts percent cytotoxicity, and the x-axis depicts concentration (nM). FIG.4 shows a graph of the cytotoxicity of RAMOS cells (B cell Lymphoma) and LP- 1 cells (Multiple Myeloma) treated with Dara-BiCE, HexaBody-CD38, daratumumab, and a BiCE negative control antibody. The y-axis depicts percent cytotoxicity, and the x-axis depicts concentration (nM). A table of receptor number indicates that LP-1 cells have a high level of CD38 expression. FIG. 5 shows a heat map of protein levels and max cell lysis of cell lines treated with Dara-IgG BiCE, Daratumumab, or the CD38 hexabody. FIG. 6 shows a graph of cytotoxicity of DOHH2 cells treated with CM91-751; CM1253-1255 (Felzartamab); CM1267-1269 (mezagitamab); CM1781-1782 (CM313); CM1781-1783 (CM313 + C1q binder); daratumumamb; CM220-221 (CD38 Hexabody); or isatuximab. The y-axis depicts percent cytotoxicity, and the x-axis depicts concentration (nM). FIG. 7 shows a graph of cytotoxicity of WSU cells treated with CM1232-1234 (daratumumamb 005); CM1233-1234; CM1232-1235; CM1232-1236; CM1232-1237; CM1239-1241; CM1240-1241; CM1239-1242; CM1239-1243; CM1239-1244; CM1246- 1248; CM1247-1248. The y-axis depicts percent cytotoxicity, and the x-axis depicts concentration (nM). FIG.8 shows a graph of the cytotoxic effects of additional CD38-targeting polypeptide constructs. The y-axis depicts percent cytotoxicity, and the x-axis depicts concentration (nM). FIG.9 shows a graphs of the cytotoxic effects of C1q and CD38-targeting polypeptide constructs (BiCE constructs) on WSU cells relative to their parental monoclonal antibody. The y-axis depicts percent cytotoxicity, and the x-axis depicts concentration (nM). FIG.10 shows graphs of the level of plasmablast and NK cell depletion in healthy bone marrow from donor treated with Dara-BiCE. The y-axis depicts percent cells from live, and the x-axis depicts concentration (nM).
FIG. 11 shows a graph of plasma cell killing in human bone marrow samples treated with Dara-BiCE. Samples were from bone marrow samples from newly diagnosed multiple myeloma (MM) patients, a relapsing patient, and a patient with progressive MM The y-axis depicts percent plasma cell killing, and the x-axis depicts the construct. FIG. 12A-FIG.12D show graphs of an ADCC bioassay of cells treated with CD38- targeting constructs provided herein. The y-axis depicts percent lysis, and the x-axis depicts the concentration. FIG. 13 shows a graph of C4a concentration in response to 003 Bice; MORO3080 BiCEs; meza BiCEs; and 024 BiCEs relative to controls. The y-axis depicts C4a concentration (micrograms per milliliter), and the x-axis depicts the construct. FIG.14 shows a graph of SRG rat tumor size over time in rats treated with palivizumab (20mg/kg) or varying concentrations of Dara-BiCE. DETAILED DESCRIPTION CD38 is a transmembrane protein that is expressed on the surface of B lymphocytes. It is a well-known target for antibody-based therapies, in particular in multiple myeloma. In such therapies, as mentioned by Leleu et al., Annals of Hematology (2022) 101:2123–2137, known anti-CD38 antibodies such as daratumumab and isatuximab “exert their therapeutic effects via direct effector mechanisms on MM cells (i.e., CD38 enzymatic inhibition, direct induction of apoptosis) and through Fc-dependent immune mechanisms, including antibody-dependent cellular cytotoxicity (ADCC), complement-directed cytotoxicity (CDC), and antibody-directed cellular phagocytosis (ADCP).” Reference is for example also made to Gozetti et al., Human vaccines & immunotherapeutics, 2022, Vol.18, No.5; to the references cited by Leleu et al. and by Gozetti et al; as well as to the further references cited herein. Some of the known antibodies against CD38 that have been approved or that are in various stages of research or development include daratumumab, isatuximab, the anti-CD38 antibodies known as Clone 003 and Clone 024 (GenMab; see also WO2006/099875), the anti- CD38 antibodies known as Moro3080, Moro 3087 and Moro 3088 (Morphosys), CM313, mezagitamab, felzartamab and the single domain antibody against CD38 known as MU1053. Other antibodies against CD38 and the indications for which they are being used and/or developed will be clear to the skilled person. As mentioned herein, these known antibodies against CD38, as well as the antibodies, antibody constructs and compositions described herein, can be used in the prevention and/or treatment of CD38-related diseases and disorders (as defined herein). Such CD38-related
diseases and disorders may in particular but without limitation be one of the following diseases or disorders: Multiple myeloma; Other types of cancer for which known antibodies against CD38 are being used, being developed or have been suggested/validated as a treatment options, such as: Al amyloidosis, T-cell lymphoma and leukemia, lymphoma, Acute myeloid leukemia; Chronic Lymphocytic Leukemia (CLL); small cell lung cancer, or triple-negative breast cancer. other diseases and disorders that can be prevented or treated by targeting CD38 or CD38 expressing cells in the body of subject to be treated, such as: kidney diseases (see for example Chen et al., Front. Immunol., 10 May 2024, Vol.15); autoimmune and inflammatory diseases (see for example Ye et al., Autoimmunity Reviews, Volume 22, Issue 4, April 2023, 103289);; Hemophilia, Antibody-mediated organ rejection, Platelet Transfusion Primary Antiphospholipid Syndrome, Hemolytic Anemia, Immune Thrombocytopenia, Systemic Lupus Erythematosus, Lupus Nephritis, Immunoglobulin A (IgA) Nephropathy, Nephrotic Syndrome, Membranoproliferative Glomerulonephritis, Proliferative Glomerulonephritis With Monoclonal IgG Deposits, Neuromyelitis Optica Spectrum Disorder|NMO Spectrum Disorder, POEMS Syndrome, myasthenia gravis, Disease or disorders related to allergy (see for example Blankestijn et al., the journal of allergy and clinical immunology Volume 139, Issue 5P1677-1678.E3May 2017) As mentioned herein, it is assumed that the antibodies of the invention will generally assert their favorable effect upon the body of a subject to be treated by direct effector mechanisms on MM cells (i.e., CD38 enzymatic inhibition, direct induction of apoptosis) and through Fc-dependent immune mechanisms (see also Leleu et al., supra). However, it should be noted that the invention in its broadest sense is not limited to any particular explanation, hypothesis or mechanism-of-action. In particular, it is not excluded that the antibodies of the invention may exert their beneficial influence upon the body of a subject treated with an antibody of the invention via any suitable mechanism-of-action, including but not limited to any mechanism-of-action known per se for one or more of the known antibodies against CD38 referred to herein. While a number of the known antibodies against CD38 have been successfully applied to the prevention and treatment of diseases and disorders in human subject, clinical practice has shown there is a continuous need for new antibodies and antibody constructs against CD38
(and in particular improved antibodies against CD38) that can be used in the prevention and treatment of diseases and disorders in human subjects. The present invention provides such antibodies and antibody constructs. Generally, the invention provides an antibody or antibody construct comprising two antibody heavy chains and two antibody light chains (which antibody heavy chains and antibody light chains form an antibody that is directed against CD38), which antibody or antibody construct suitably comprises two single domain antibodies that are directed against C1q (in accordance with the co-pending International applications referred to herein, such single domain antibodies against C1q are also referred to herein as “C1q binders”). The invention in particular provides such an antibody or antibody construct in which the antibody heavy chains and antibody light chains form an antibody that is directed against the same epitope on CD38 as daratumumab. In a further aspect, the invention provides such an antibody or antibody construct in which the two VH domains that are present in the heavy chains contain the same CDRs as the VH domain that is present in daratumumab and in which the two VL domains that are present in the light chains contain the same CDRs as the VL domain that is present in daratumumab (in which said CDRs are the CDRs according to Kabat). In a further aspect, the invention provides such an antibody or antibody construct in which the two VH domains that are present in the heavy chains have the same amino acid sequence as the VH domain that is present in daratumumab and in which the two VL domains that are present in the light chains have the same amino acid sequence as the VL domain that is present in daratumumab. In a preferred aspect, the antibodies of the invention are as described in the co-pending International applications referred to herein. Accordingly, as described in these co-pending International applications, in the antibodies of the invention, most preferably and usually: the two heavy chains will have the same amino acid sequence and the two light chains will have the same amino acid sequence; the two C1q binders that are present in the antibodies of the invention will have the same amino acid sequence; when one of the C1q binders is fused or linked to one of the heavy chains, then the other C1q binder will be fused or linked to the other heavy chain; when one of the C1q binders is fused or linked to one of the light chains, then the other C1q binder will be fused or linked to the other light chain;
when one of the C1q binders is fused or linked to the N-terminus of a heavy or light chain, respectively, then the other C1q binder will also be fused or linked to N-terminus of the other heavy or light chain, respectively; when one of the C1q binders is fused or linked to the C-terminus of a heavy or light chain, respectively, then the other C1q binder will also be fused or linked to C-terminus of the other heavy or light chain, respectively; and the C1q binder can be as generally described in WO2019/238674, in WO2020/167919 or in the co-pending International applications, but is preferably a C1q binder that is as generally described in the co-pending International applications and that has an affinity for C1q of between 10 nanoMolar (nM) to about 2 microMolar (μM), as determined by biolayer interferometry, in particular of about 0.1 μM to about 2 μM, as determined by biolayer interferometry; the C1q binder is preferably a variant of Nb75 or Nb78, and in particular a humanized variant of Nb75 or Nb78 (as described in the co-pending International applications); when two elements of a construct are “fused or linked” (as further defined in the Co- pending International applications), any suitable linker known per se may be used for this purpose (for which reference is again made to the Co-pending International applications). In a particular aspect, the invention relates to an antibody of the invention in which the one of the C1q binders is linked to the C-terminus of one of the light chains, and the other C1q binder is linked to the C-terminus of the other light chain. Also, in the antibodies of the invention according to this aspect, the C1q binder is preferably a humanized variant of Nb78 with an affinity for C1q of between 10 nanoMolar (nM) to about 2 microMolar (μM), as determined by biolayer interferometry, in particular of about 0.1 μM to about 2 μM, as determined by biolayer interferometry. In a particularly preferred aspect, the invention relates to an antibody of the invention in which the two heavy chains both (essentially) comprise the amino acid sequence of SEQ ID NO:1 (not taking into account any C1q binders that maybe fused or linked to said heavy chains). SEQ ID NO:1 is the amino acid sequence of the heavy chain of daratumumab. EVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSAIS GSGGGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCAKDKILW FGEPVFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFP EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH
KPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 1); and in which the two light chains both (essentially) comprise the amino acid sequence of SEQ ID NO:2 (not taking into account any C1q binders that maybe fused or linked to said light chains). SEQ ID NO:2 is the amino acid sequence of the light chain of daratumumab. EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASN RATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GEC (SEQ ID NO: 2). An antibody of the invention may comprise a naturally occurring Fc portion or a non- naturally occurring Fc portion (such as, for example and without limitation, an Fc portion that has been derived from a naturally occurring Fc portion but that (suitably) carries, within its sequence, one or more of the mutations referred to herein for such non-naturally occurring Fc portions). Some specific but non-limiting examples of mutations that can be present in the Fc portion of (the heavy chains that are present in) an antibody of the invention are described herein and/or listed in Table B; and for illustration purposes only Table C gives examples of such non-naturally occurring Fc regions (using the Fc of the anti-CD20 antibody 11B8 as a reference sequence, again for illustration purposes only). Such mutations may also be used in a suitable combination, as will be clear to the skilled person (who on the basis of the disclosure herein will also be able to make essentially the same or similar mutations in another naturally occurring Fc region such as the Fc region that is present in the sequence of SEQ ID NO:1). For example and without limitation, such mutations or suitable combination of mutations may (i) alter (i.e. increase or decrease) the half-life of the antibody of the invention; (ii) alter (i.e. increase or decrease) the binding/affinity of the antibody to Fc gamma receptors; and/or (iii) alter (i.e. increase or decrease) the binding/affinity of the Fc region and/or the antibody of the invention for C1q (with but preferably essentially without affecting the affinity for C1q of the
C1q binder(s) present in the antibody of the invention). These and other suitable mutations will be clear to the skilled person based on the disclosure herein. In one aspect, the invention relates to an antibody of the invention (as further described herein) that contains a non-naturally occurring Fc region, which non-naturally occurring Fc region contains at least one mutation that alter (i.e. increase or decrease, but in particular decrease or essentially removed) the binding/affinity of the antibody to Fc gamma receptors. In particular, the invention relates to an antibody of the invention (as further described herein) that contains a non-naturally occurring Fc region, which non-naturally occurring Fc region contains at least one mutation that alter (i.e. increase or decrease, but in particular decrease or essentially removed) the binding/affinity of the antibody to Fc gamma receptors, where such mutations essentially do not affect the affinity of the antibody of the invention for C1q. Again, suitable mutations or combinations of mutations will be clear to the skilled person based on the disclosure herein or can easily be determined (optionally after a limited degree of testing) by the skilled person, and for example include K320E, Q386R) deletion of G236 and/or K326W, E333S. Some specific but non-limiting examples of antibodies against CD38 are listed in Table A below. In a specifically preferred aspect, the invention relates to an antibody of the invention in which the two heavy chains both (essentially) comprise the amino acid sequence of SEQ ID NO: 1 (and are not fused or linked to a C1q binder) and in which the two light chains both (essentially) comprise the amino acid sequence of SEQ ID NO: 3 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASN RATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GECGGGGSGGGGSQVQLVESGGGSVQPGGSLRLSCTASGWTFRDSAYNLGW FRQAPGQEREAVAAISWRGGSTYYADSVKGRFTISRDNAKNTVTLQMNNLK PEDTAIYYCAADASARAALYSTGYEYDHWGQGTQVTVSS (SEQ ID NO:3) SEQ ID NO:3 comprises the light chain of daratumumab, which is fused at its C-terminus, via a GGGGSGGGGS linker (SEQ ID NO: 4), to the C1q binder of SEQ ID NO:5. SEQ ID NO:
5 is a humanized variant of Nb78 with reduced affinity for C1q, as described in the co-pending International applications. QVQLVESGGGSVQPGGSLRLSCTASGWTFRDSAYNLGWFRQAPGQEREAVA AISWRGGSTYYADSVKGRFTISRDNAKNTVTLQMNNLKPEDTAIYYCAADAS ARAALYSTGYEYDHWGQGTQVTVSS (SEQ ID NO: 5) The antibodies provided by the invention (and in particular those that comprise a C1q binders with affinity for C1q of between 10 nanoMolar (nM) to about 2 microMolar (μM), as determined by biolayer interferometry, in particular of about 0.1 μM to about 2 μM, as determined by biolayer interferometry) have a number of advantages over known antibodies against CD38, such as rituximab and daratumumab. These generally include the following (individually and in combination): improved ability to induce CDC, compared to an antibody that does not comprise a C1q binder as described herein (and in particular, compared to daratumumab); the advantages described in the co-pending International applications associated with the use of an antibody format that comprises two single domain antibodies against C1q (as compared to an antibody that does not contain any C1q binder or only a single C1q binder); the advantages described in the co-pending International applications associated with the use of an antibody format that comprises a single domain antibody against C1q (and in particular two such C1q binders) having an affinity for C1q within the ranges specified above (as compared to an antibody format that comprises one or two C1q binders with “better”/”higher” affinity for C1q);improved ability to “activate complement on” a cell that expresses CD38 to which the antigen binding domain is directed (meaning: to trigger/recruit the complement system to mount an immune response or other reaction against such cell) compared to the same antibody without the C1q binder(s); and/or no requirement for hexamerization upon antigen binding which is needed for efficient complement activation by IgG as described by Diebolder at al, Science. 2014 Mar 14;343(6176):1260–1263. This means that more epitopes can be targeted; and/or potential to recruit more than one C1q resulting in more potent complement activation as a C1q bispecific construct as described herein can – for example - have 3 C1q binding sites whereas an IgG has 1;
The antibodies may in addition have one or more of the following advantages (again, compared to the same antibody without the C1q binder), depending on the Fc portion that is present in the specific antibody and in particular on the Fc mutation(s) that may be present in such Fc portion: the ability to activate complement on a cell that expresses CD38 (i.e. on its surface), without binding to Fc-gamma receptors on immune cells thereby preventing effector mechanisms ADCC and ADCP (in other words, provide a degree of selectivity towards triggering CDC); or the ability to activate complement on a cell that expresses CD38 (i.e. on its surface), without preventing binding to Fc-gamma receptors on immune cells thereby allowing for additional secondary effector mechanisms including ADCC and ADCP. In another aspect, the invention relates to a nucleic acid that encodes an antibody of the invention (as mentioned, also referred to herein as a “nucleic acid of the invention”). Such a nucleic acid of the invention may for example be in the form of a genetic construct, as will be clear to the person skilled in the art and as described on pages 131-134 of WO 08/020079 (incorporated herein by reference). Such genetic constructs generally comprise at least one nucleic acid of the invention that is optionally linked to one or more elements of genetic constructs known per se, such as for example one or more suitable regulatory elements (such as a suitable promoter(s), enhancer(s), terminator(s), etc.) and the further elements of genetic constructs known per se. For example, such a genetic construct may be in a form known per se for the expression or production of the known antibodies against CD38 referred to herein and/or in a suitable form as described in WO2019/238674, in WO2020/167919 or in the co- pending International applications. In a further aspect, the invention relates to a host or host cell that expresses and/or produces (or that under suitable circumstances is capable of expressing and/or producing) an antibody of the invention; and/or that contains a nucleic acid of the invention. Suitable host cells and cell lines will be clear to the skilled person and may, for example and without limitation, include the cells and cell lines known per se that are used for the expression or production of the known antibodies against CD38 referred to herein, and in particular be mammalian cells (or a mammalian cell line) that are suitable for expressing/producing the antibodies of the invention and/or suitable host cells or cell lines as described in WO2019/238674, in WO2020/167919 or in the co-pending International applications.
The invention also relates to a method of expressing or producing an antibody of the invention, which method at least comprises the step of maintaining a host or host cell that expresses and/or produces (or that under suitable circumstances is capable of expressing and/or producing) an antibody of the invention (and/or that contains a nucleic acid of the invention) under conditions such that said host cell expresses/produces said antibody of the invention. Suitable conditions will be clear to the skilled person based on the disclosure herein and may depend on the particular host cell or cell line used. Reference is again for example made to WO2019/238674, WO2020/167919 and the co-pending International applications. The invention further relates to a product or composition containing or comprising at least one antibody of the invention and/or at least one nucleic acid of the invention, and optionally one or more further components of such compositions known per se, i.e. depending on the intended use of the composition. Such a product or composition may for example be a pharmaceutical composition (as described herein), a veterinary composition or a product or composition for diagnostic use (as also described herein). Some preferred but non-limiting examples of such products or compositions will become clear from the further description herein. The antibodies of the invention can also be administered using gene therapy methods of delivery (including suitable vaccination methods such as mRNA or DNA vaccination methods) such that, upon such administration, an antibody of the invention is suitably expressed/formed in the body of the subject to be treated. Suitable gene therapy vectors, mRNA vaccines and DNA vaccines and suitable methods of administering the same will be clear to the skilled person. Reference is for example made to U.S. Patent No. 5,399,346, which is incorporated by reference in its entirety. Using a gene therapy method of delivery, primary cells transfected with the gene encoding an antibody of the invention can additionally be transfected with tissue specific promoters to target specific organs, tissue, grafts, tumors, or cells and can additionally be transfected with signal and stabilization sequences for subcellularly localized expression. Generally, for pharmaceutical use, the antibodies of the invention may be formulated as a pharmaceutical preparation or compositions comprising at least one antibody of the invention and at least one pharmaceutically acceptable carrier, diluent or excipient and/or adjuvant, and optionally one or more further pharmaceutically active polypeptides and/or compounds. By means of non-limiting examples, such a formulation may be in a form suitable for oral administration, for parenteral administration (such as by intravenous, intramuscular or
subcutaneous injection or intravenous infusion), for topical administration, for administration by inhalation, by a skin patch, by an implant, by a suppository, etc.. Such suitable administration forms - which may be solid, semi-solid or liquid, depending on the manner of administration - as well as methods and carriers for use in the preparation thereof, will be clear to the skilled person based on the disclosure herein, and may for example and without limitation, include formulations (and components and constituents of formulations) known per se that can be used for formulating the known antibodies against CD38 referred to herein and/or the formulations described in WO2019/238674, WO2020/167919 or in the co-pending International applications. Thus, in a further aspect, the invention relates to a pharmaceutical composition that contains at least one antibody of the invention and at least one suitable carrier, diluent or excipient (i.e. suitable for pharmaceutical use), and optionally one or more further active substances. Generally, the antibodies of the invention can be formulated and administered in any suitable manner known per se, for which reference is for example made to the general background art cited above (and in particular to WO 04/041862, WO 04/041863, WO 04/041865, WO 04/041867 and WO 08/020079) as well as to the standard handbooks, such as Remington’s Pharmaceutical Sciences, 18th Ed., Mack Publishing Company, USA (1990), Remington, the Science and Practice of Pharmacy, 21th Edition, Lippincott Williams and Wilkins (2005); or the Handbook of Therapeutic Antibodies (S. Dubel, Ed.), Wiley, Weinheim, 2007 (see for example pages 252-255). These may for example and without limitation, include means and methods for administration known per se for the administration of the known antibodies against CD38 referred to herein as well as means and methods for administration as described in WO2019/238674, WO2020/167919 or in the co-pending International applications. For example, the antibodies in may be formulated and administered in any manner known per se for conventional antibodies (including the known anti-CD38 antibodies mentioned herein). Such formulations and methods for preparing the same will be clear to the skilled person, and for example include preparations suitable for parenteral administration (for example intravenous, intraperitoneal, subcutaneous, intramuscular, intraluminal, intra-arterial or intrathecal administration) or for topical (i.e. transdermal or intradermal) administration. Reference is again also made to WO2019/238674, WO2020/167919 and the co-pending International applications.
Preparations for parenteral administration may for example be sterile solutions, suspensions, dispersions or emulsions that are suitable for infusion or injection. Suitable carriers or diluents for such preparations for example include, without limitation, those mentioned in WO2019/238674, WO2020/167919 and the co-pending International applications. Usually, aqueous solutions or suspensions will be preferred. As will be clear to the skilled person and/or the treating physician, the amount(s) of the antibodies of the invention required for use in treatment (i.e. the dose(s) and dose regimen applied in such treatment) will vary not only with the particular antibody of the invention used, but also with the route of administration, the nature of the condition being treated and the age and condition of the patient and will be ultimately at the discretion of the attendant physician or clinician, who will be able to choose an appropriate dose regimen taking into account the considerations referred to herein. The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day. The sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations. An administration regimen could include long-term, daily treatment. By “long-term” (or “chronic”) is meant at least two weeks and preferably, several weeks, months, or years of duration. Necessary modifications in this dosage range may be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein. See Remington’s Pharmaceutical Sciences (Martin, E.W., ed.4), Mack Publishing Co., Easton, PA. The dosage can also be adjusted by the individual physician in the event of any complication. In another aspect, the invention relates to a method for the prevention and/or treatment of at least CD38-related disease or disorder (as defined herein), said method comprising administering, to a subject in need thereof, a pharmaceutically active amount of an antibody of the invention and/or of a pharmaceutical composition comprising the same (i.e. in one or more suitable doses and according to a suitable dosage regimen, as further described herein). In another aspect, the invention relates to a method for increasing the complement- dependent cytotoxicity towards a CD38 expressing cell that is exerted by the body of a subject that is in need thereof, which method comprises administering, to said subject, a pharmaceutically active amount of an antibody of the invention or a pharmaceutical composition comprising the same. In the context of the present invention, the term “prevention and/or treatment” not only comprises preventing and/or treating the disease, but also generally comprises preventing the
onset of the disease, slowing or reversing the progress of disease, preventing or slowing the onset of one or more symptoms associated with the disease, reducing and/or alleviating one or more symptoms associated with the disease, reducing the severity and/or the duration of the disease and/or of any symptoms associated therewith and/or preventing a further increase in the severity of the disease and/or of any symptoms associated therewith, preventing, reducing or reversing any physiological damage caused by the disease, and generally any pharmacological action that is beneficial to the patient being treated. The subject to be treated may be any warm-blooded animal, but is in particular a mammal, and more in particular a human being. As will be clear to the skilled person, the subject to be treated will in particular be a person suffering from, or at risk of, the diseases and disorders mentioned herein. In another aspect, the invention relates to a method for immunotherapy, which method comprises administering, to a subject suffering from or at risk of a CD38-related disease or disorder, a pharmaceutically active amount of an antibody of the invention or a pharmaceutical composition comprising the same. As mentioned herein, in the practice of the invention, the antibody or composition of the invention will generally be administered to the subject to be treated according to a regime of treatment that is suitable for preventing and/or treating the disease or disorder to be prevented or treated. The clinician will generally be able to determine a suitable treatment regimen, depending on factors such as the disease or disorder to be prevented or treated, the severity of the disease to be treated and/or the severity of the symptoms thereof, the specific antibody of the invention used, the specific route of administration and pharmaceutical formulation or composition to be used, the age, gender, weight, diet, general condition of the patient, and similar factors well known to the clinician. Generally, the treatment regimen will comprise the administration of one or more antibodies of the invention, or of one or more compositions comprising the same, in one or more pharmaceutically effective amounts or doses. The specific amount(s) or doses to administered can be determined by the clinician, again based on the factors cited herein. Generally, for the prevention and/or treatment of the diseases and disorders mentioned herein and depending on the specific disease or disorder to be treated, the potency of the specific antibody of the invention, the specific route of administration and the specific pharmaceutical formulation or composition used, the antibodies of the invention will generally be administered in an amount between 1 gram and 0.01 microgram per kg body weight per day, preferably between 0.1 gram and 0.1 microgram per kg body weight per day, such as about 1,
10, 100 or 1000 microgram per kg body weight per day, either continuously (e.g. by infusion), as a single daily dose or as multiple divided doses during the day. The clinician will generally be able to determine a suitable daily dose, depending on the factors mentioned herein. It will also be clear that in specific cases, the clinician may choose to deviate from these amounts, for example on the basis of the factors cited above and his expert judgment. Generally, some guidance on the amounts to be administered can be obtained from the amounts usually administered for comparable conventional antibodies or antibody fragments against the same target administered via essentially the same route, taking into account however differences in affinity/avidity, efficacy, biodistribution, half-life and similar factors well known to the skilled person. Usually, in the above method, a single antibody of the invention will be used. It is however within the scope of the invention to use two or more antibodies of the invention in combination. The antibodies of the invention may also be used in combination with one or more further pharmaceutically active compounds or principles, i.e. as a combined treatment regimen, which may or may not lead to a synergistic effect. Again, the clinician will be able to select such further compounds or principles, as well as a suitable combined treatment regimen, based on the factors cited above and his expert judgement. In particular, the antibodies of the invention may be used in combination with other pharmaceutically active compounds or principles that are or can be used for the prevention and/or treatment of the diseases and disorders cited herein, as a result of which a synergistic effect may or may not be obtained. Examples of such compounds and principles, as well as routes, methods and pharmaceutical formulations or compositions for administering them will be clear to the clinician, and for example include the active compounds or principles that are or can be used in combination with one of the known antibodies against CD38 referred to herein (e.g. according to a suitable combined treatment regimen, as will be able to be determined by the treating clinician). When two or more substances or principles are to be used as part of a combined treatment regimen, they can be administered via the same route of administration or via different routes of administration, at essentially the same time or at different times (e.g. essentially simultaneously, consecutively, or according to an alternating regime). When the substances or principles are to be administered simultaneously via the same route of administration, they may be administered as different pharmaceutical formulations or
compositions or part of a combined pharmaceutical formulation or composition, as will be clear to the skilled person. Also, when two or more active substances or principles are to be used as part of a combined treatment regimen, each of the substances or principles may be administered in the same amount and according to the same regimen as used when the compound or principle is used on its own, and such combined use may or may not lead to a synergistic effect. However, when the combined use of the two or more active substances or principles leads to a synergistic effect, it may also be possible to reduce the amount of one, more or all of the substances or principles to be administered, while still achieving the desired therapeutic action. This may for example be useful for avoiding, limiting or reducing any unwanted side-effects that are associated with the use of one or more of the substances or principles when they are used in their usual amounts, while still obtaining the desired pharmaceutical or therapeutic effect. The effectiveness of the treatment regimen used according to the invention may be determined and/or followed in any manner known per se for the disease or disorder involved, as will be clear to the clinician. Suitable methods and techniques will be clear to the skilled person and will often generally comprise a step of determining the level(s) of the relevant CD38 expressing cell(s) in the body of the subject to be treated (or in a suitable biological sample obtained from such subject). and/or determining any changes in such levels. Such methods and techniques may for example include the methods and techniques commonly used for determining the effectiveness and/or course of a treatment with one of the known antibodies against CD38 referred to herein. The clinician will also be able, where appropriate and on a case-by-case basis, to change or modify a particular treatment regimen, so as to achieve the desired therapeutic effect, to avoid, limit or reduce unwanted side-effects, and/or to achieve an appropriate balance between achieving the desired therapeutic effect on the one hand and avoiding, limiting or reducing undesired side effects on the other hand. Generally, the treatment regimen will be followed until the desired therapeutic effect is achieved and/or for as long as the desired therapeutic effect is to be maintained. Again, this can be determined by the clinician. In another aspect, the invention relates to the use of an antibody of the invention in the preparation of a pharmaceutical composition for prevention and/or treatment of at least one CD38-related disease or disorder (as defined herein); and/or for use in one or more of the methods of treatment mentioned herein. The invention further relates to an antibody of the invention (or a pharmaceutical composition comprising the same) for the prevention and/or
treatment of at least one CD38-related disease or disorder (as defined herein). The invention also relates to a method of preventing and/or treating a CD38-related disease or disorder (as defined herein), which method comprises administering, to a subject in need thereof, of an antibody of the invention or a pharmaceutical composition comprising the same (again, according to a suitable route of administration and a suitable dosage regimen, as further described herein). Further uses of the antibodies and compositions of the invention will be clear to the skilled person based on the disclosure herein. For example and without limitation, the antibodies of the invention can be linked to a suitable detectable label and used as markers to detect or determine (qualitatively or quantitatively) the presence of CD38 or CD38 expressing cells, either in vitro, ex vivo or in vivo; and/or to determine whether the antibodies of the invention are capable of bringing together CD38-expressing cells and C1q (i.e. in order to initiate complement-dependent cytotoxicity by the complement system ). The invention will now be further described by means of the following non-limiting examples and figures. EXAMPLES EXAMPLE 1. Generation and Evaluation of Polypeptide Constructs Comprising C1q Complement Factor-Engaging Domains. Generation of Polypeptide Constructs A polypeptide construct with a complement factor-engaging domain at the C-terminus of an antibody light chain was designed in silico together with a corresponding heavy chain gene. Several affinity variants of the polypeptide constructs comprising C1q complement factor- engaging domains as disclosed herein were designed and tested in this format. Genes we synthesized and cloned into a pCDNA.3.1 vector which was used for transfection of Expi293 cells. Expi293 cells were transfected by mixing OPTI-MEM, DNA and PEI Max which was added to cells. 18h post transfection, each of the transfections were added Valproic acid, sodium propionate, and glucose to enhance the transfection efficacy. Cells were incubated at 37 °C with 8 % CO2. After 6 days, the supernatant was harvest by centrifugation at 200 g for 10 min at room temperature followed by a centrifugation at 4000 g for 30 min at room temperature. The supernatant was added 1 M Tris pH 8 to final concentration of 50 mM and mixed with protein A beads equilibrated in PBS. Beads were incubated on rotation at 4 °C for 1 h and loaded on a gravity column. Beads were washed with 15 column volumes of PBS with 350 mM NaCl added and eluted with 50 mM glycine pH 2.7 into tubes containing 1M TRIS pH 8. Polypeptide purity was evaluated by SDS-PAGE and concentrated to
l. Samples were centrifuged for 10 minutes at 4000 rpm at 4 °C and loaded on a Superdex 200 Increase 10/300 GL column in PBS. Eluted fractions containing polypeptide constructs were analysed by SDS-PAGE and fractions containing the polypeptide constructs were pooled. EXAMPLE 2: CYTOTOXICITY OF CD38 TARGETING CONSTRUCTS The following polypeptide constructs were manufactured in the table below and as shown in FIG. 1 according to the methods in EXAMPLE 1. TABLE 1: CD-38 Constructs Name SEQ ID NOS: Targets Dara-BiCE C1q binding moiety: SEQ ID NO: 5 C1q Heavy and Light chain: SEQ ID CD-38 NOS: 1 and 2 HexaBody-CD38 -- CD-38 Daratumumab Heavy and Light chain: SEQ ID CD-38 NOS: 71 and 72 BiCE negative control -- C1q Multiple tumor cell lines were obtain with varying levels of CD38 expression. Each cell line in Table 2 was tested in response to treatment with the polypeptide constructs in Table 1 and maximum cell lysis was quantified (FIG.2). CD38-IgG BiCE was superior to CD38- Hexabody and Daratumumab across a broad cell line panel for maximum cancer cell lysis. TABLE 2: Cell Lines in FIG. 2. Cell line Tumor Type CD38 CD55 CD46 Expression Expression Expression WSU-DLCL2 B cell 49712 5334 13770 lymphoma DOOH-2 B cell 76131 13844 11368 lymphoma RAMOS B cell 99777 4307 12174 lymphoma Granta-519 B-cell 17401 11003 18030 lymphoma
RS4; 11 Acute 42676 8292 15682 lymphoblastic leukemia SUP-B15 Acute 39047 1562 13274 lymphoblastic leukemia SEM Acute 30772 1852 19572 lymphoblastic leukemia Mono-mac-6 Acute 18383 4783 13005 monocytic leukemia OPM-2 Multiple 36224 10890 20522 myeloma LP-1 Multiple 191214 1997 26129 myeloma A representative graph of complement dependent cytotoxicity observed in WSU- DLCL2 cells is shown in FIG.3. All cell lines treated with Dara-BiCE had increased levels of cytotoxicity relative to daratumumab and the negative control. Similar activity of Dara-BiCE and HexaBody-CD38 were observed in cell lines with very high levels of CD38 (FIG. 4). Protein levels and max lysis of the tested cell lines were quantified (FIG. 5). Next, CD38 mAbs and CD38 BiCE versions listed in Table 3 below were evaluated in a CDC Assay of DOHH2 cells, a human B cell lymphoma cell line. CytoToxGlo™ (Promega, Madison, WI, USA) was used to evaluate cell cytotoxicity according to manufacturer’s instructions using 100,000 cells/well and 10% NHS. TABLE 3. CD38-targeting polypeptide and antibody constructs. Name Composition SEQ ID NOs: daratumumab Hc SEQ ID NO: 1 Dara Fab Lc SEQ ID NO: 9 10GS GS linker CM91-751 hNb78 (M33A,T102A) SEQ ID NO: 21 Felzartamab (MORO3087) Hc SEQ ID NO: 10 CM1253-1255 MORO3087 Lc SEQ ID NO: 11 Mezagitamab Hc SEQ ID NO: 12 CM1267-1269 Mezagitamab Lc SEQ ID NO: 13
CM313 Hc SEQ ID NO: 33 CM1781-1782 CM313 Lc SEQ ID NO: 34 CM313 Hc SEQ ID NO: 33 CM313 Lc SEQ ID NO: 34 5GS GS linker CM1781-1783 hNb78 (M33A,T102A) SEQ ID NO: 21 SEQ ID NO: 1 and SEQ ID Daratumumab Daratumumab NO: 2 CD38 Hc hexabody -- CM220-221 CD38 Lc -- SEQ ID NO: 31 and SEQ ID Isatuximab Isatuximab NO: 32 Lc: light chain Hc: heavy chain GS: GS linker G: G linker See also Table A for sequences The IgG-BiCEs polypeptide constructs, the CD38-Hexabody, and felzartamab induced cytotoxicity of DOHH2 cells. No CDC was observed for cells treated with Mezagitamab, CM313, Daratumumab or Isatuximab, while Felzartamab only induced CDC at high concentrations (FIG.4). The CD38 BiCE showed high efficacy and potency, which is also better compared to CD38-Hexabody which display both lower max lysis and higher EC50 value (See Table 5). TABLE 5. EC50 Values Construct Composition Max EC50 CM91-751 Dara (005) Hc and Lc 52.1 0.74 CM1253-1255 felzartamab 33.4 NA CM1267-1269 Mezagitamab 0.097 NA CM1781-1782 CM313 mAb 1.84 NA CM1781-1783 CM313 T102A 52.9 0.06 Daratumumab Daratumumab 0.623 NA CM220-221 CD38-Hexabody 40.2 0.59 Isatuximab Isatuximab 1.2 0.15 Lc: light chain Hc: heavy chain See also Table A for sequences Additional CD38 mAbs in Table 6 were evaluated in different IgG BiCE-formats as
shown in FIG. 6-FIG. 9. The sequences for the polypeptide constructs are provided below. The structures of each construct are provided in FIG.1. In some cases, the C1q binder is hNb78 [M33A, T102A] having a sequence of: QVQLVESGGGSVQPGGSLRLSCTASGWTFRDSAYNLGWFRQAPGQEREAVAAISWR GGSTYYADSVKGRFTISRDNAKNTVTLQMNNLKPEDTAIYYCAADASARAALYSTG YEYDHWGQGTQVTVSS (SEQ ID NO: 21); or Nb75 [T53G] QVQLVETGGGLVQAGGSLRLSCAASGRTFNNDVMAWFRQAPGTEREFVALIGAGG GTHYADSVKGRFVISRDNDKNMAYLQMNSLKSEDTAIYYCGADENPPGWPSRWSS AYDYWGQGTQVTVSS (SEQ ID NO: 22) In some cases, the polypeptide constructs with a C1q complement factor-engaging domain at the C-terminus of the heavy-chain (HC) of an IgG region of a Genmab CD38- targeting antibody clone 003 were generated as described herein. The clone 003 sequence comprises the following heavy and light chain sequences below. Clone 003 Light chain: DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASS LQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPRTFGQGTKVEIKRTVA APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 23) Clone 003 Heavy chain: QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAFSWVRQAPGQGLEWMGR VIPFLGIANSAQKFQGRVTITADKSTSTAYMDLSSLRSEDTAVYYCARDDIAALGPFD YWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K (SEQ ID NO: 24) TABLE 6. Exemplary CD38 monoclonal antibodies and polypeptide constructs. Name Composition SEQ ID NO: CM1232-1234 Daratumumab (Dara 005) Hc and Lc SEQ ID NO: 1 and SEQ ID NO: 2
Nb75 (T53G)-10GS- Dara 005 Hc + Dara SEQ ID NO: 22; SEQ ID NO: 6 and CM1233-1234 005 Lc SEQ ID NO: 2 Dara (005) Hc + Dara modified Lc - 4G - SEQ ID NO: 1; SEQ ID NO: 7; and CM1232-1235 hNb78(M33A,T102A) SEQ ID NO: 21 Dara 005 Hc + Dara modified Lc - 5G - SEQ ID NO: 1; SEQ ID NO: 8 and CM1232-1236 hNb78(M33A,T102A) SEQ ID NO: 21 Dara 005 Hc + Nb75(T53G)-10GS- Dara SEQ ID NO: 1; SEQ ID NO: 22; and CM1232-1237 005 Lc SEQ ID NO: 2 CM1239-1241 Dara 003 Hc + Dara 003 Lc SEQ ID NO: 23; SEQ ID NO: 24 SEQ ID NO: 22; SEQ ID NO: 23; CM1240-1241 Nb75 (T53G)-10GS- 003 Hc + 003 Lc SEQ ID NO: 24 003 Hc + 003 Lc - 4G - SEQ ID NO: 23; SEQ ID NO: 24; CM1239-1242 hNb78(M33A,T102A) and SEQ ID NO: 21 003 Hc + 003 Lc - 10GS - SEQ ID NO: 23; SEQ ID NO: 24; CM1239-1243 hNb78(M33A,T102A) and SEQ ID NO: 21 SEQ ID NO: 23; SEQ ID NO: 22; CM1239-1244 003 Hc + Nb75(T53G)-10GS- 003 Lc SEQ ID NO: 24; CM1246-1248 MORO3080 Hc + MORO3080 Lc SEQ ID NO: 27; SEQ ID NO: 26 Nb75 (T53G)-10GS-MORO3080 Hc + SEQ ID NO: 22; SEQ ID NO: 25; CM1247-1248 MORO3080 Lc SEQ ID NO: 26 Lc: light chain Hc: heavy chain G: G linker See also Table A and Sequences section for sequences As shown in FIG.8 and FIG.9, MORO3080 (e.g., CM1246-1248) and daratumumab monoclonal antibodies (e.g., CM1232-1234) do not work alone. However, adding Nb75 (T53G) on the N terminus of the heavy chain of MORO3080 results in the lowest EC50 and the highest max of the tested constructs. Moreover, the addition of hNb78 (M33A, T102A) on the C terminus of the light chain of daratumumab potentiates CDC activity. The IgG BiCEs shown in FIG. 9 exhibited high CDC activity on hard to kill CD38 positive cancer cell lines such as B-cell non-Hodgkin lymphoma cell line, WSU- DLCL2. EXAMPLE 3: DEPLETION OF CD38 BONE MARROW CELLS Dara-BiCE was evaluated for killing of CD38 positive cells in a bone marrow from healthy human donors in the presence of complement (FIG.10). Dara-BiCE shows efficient complement mediated killing of plasmablasts and NK cells. Activity of Dara-BiCE and Daratumumab was evaluated on bone marrow samples obtained from newly diagnosed
multiple myeloma (MM) patients, a relapsing patient, and a patient with progressive MM. Dara-BiCE shows superior ex vivo killing of MM cells compared to Daratumumab (FIG.11). EXAMPLE 4: ANTIBODY-DEPENDENT CELLULAR CYTOTOXICITY (ADCC) ACTIVITY OF CD38 TARGETING CONSTRUCTS Antibody-dependent cellular cytotoxicity (ADCC) was evaluated by an ADCC assay using human PBMCs and Raji cells (KILR) that were treated with either Dara-BiCE, Daratumumab, of the CD38 Hexabody. Dara-BiCE exhibited comparable ADCC activity to daratumumab and HexaBody-CD38 as shown in Table 7. TABLE 7. ADCC Assay Max Lysis and EC50. Construct Max lysis % EC50 (nM) Dara-BiCE 41.1 0.010 Daratumumab 41.0 0.015 HexaBody-CD38 35.9 0.0062 A number of the various antibodies of the invention were compared for their ADCC activity with the antibodies from which they were derived as shown in FIG. 12A-FIG.12D. IgG BiCE constructs exhibited comparable ADCC activity relative to the parental mAbs (e.g., daratumumab (003) and MORO3080 as quantified in Table 8. TABLE 8: ADCC Summary. mAb/BiCE Maximum (% lysis) EC50 (nM) R squared CM1253-1255 41.7 0.015 0.96 (MORO3087/Felzartamab) CM1239-1241 (003) 40.2 0.013 0.98 CM1246-1248 39.7 0.0075 0.98 (MORO3080) CM1239-1244 (003 BiCE) 37.9 0.032 0.97 CM1253-1256 36.9 0.011 0.97 (MORO3087-BiCE) CM1267-1269 36.6 0.0059 0.97 (Mezagitamab) CM1232-1236 (Dara- 36.0 0.0035 0.93 BiCE)
CM1246-1251 35.0 0.0091 0.98 (MORO3080-BiCE) CM1232-1235x1239-1242 33.5 0.014 0.99 (Darax003-BiCE) CM1232-1234 (Dara) 32.1 0.0067 0.98 CM1267-1272 31.2 0.0064 0.96 (Mezagitamab-BiCE) CM1260-1263 24.2 3.5 0.97 (MORO3088-BICE) CM1260-1262 22.5 4.7 0.91 (MORO3088) Different PBMC donor CM1232-1234 (Dara) 28.7 0.016 0.98 CM1274-1275 (024) 26.3 0.3 0.97 EXAMPLE 5: EVALUATION OF CD38 TARGETING CONSTRUCTS ON CD38 ENZYMATIC ACTIVITY AND BINDING The effect of the various antibodies provided herein on CD38 enzyme activity were evaluated. Assays for cyclase activity and hydrolase activity of CD38 were performed. Dara- BiCE affected the CD38 enzymatic activity in a manner that was comparable to daratumumab. Next the cross reactivity of the CD38-targeting constructs provided herein was evaluated in NHP CD38-expressing cells. Daratumumab (CM1232-1234) does not bind any of the CD38 proteins examined. All other polypeptide constructs tested (CM1239-1241 (003), CM1246-1248 (MORO3080), CM1253-1255 (MORO3087), CM1260-1262 (MORO3088), CM1267-1269 (mezagitamab) and CM1274-1275 (024) bind all three CD38 proteins. MORO3080 binds strongly to cynomolgus monkey, marmoset, and rhesus monkey CD38 proteins. EXAMPLE 6: EPITOPE BINNING REVEALS COMBINATIONS FOR BIPARATOPIC CD38 IGG BICES. Epitope overlap of CD38 mAbs was evaluated using BLI and recombinant human CD38. BLI epitope binning was performed with CD38 clones: CM1232-1234, CM1239-1241, CM1246-1248, CM1253-1255, CM1260-1262, CM1267-1269, and CM1274-1275 on HIS sensors with hCD38-his. CM1239-1241 (003) binds a different epitope on CD38 than all the other tested CD38 antibodies. CM1274-1275 (024) binds another epitope on CD38 than the
other tested CD38 antibodies. The epitopes of CM1232-1234 (005), CM1246-1248 (MORO3080), CM1253-1255 (MORO3087), and CM1267-1269 (Mezagitamab) seem to overlap on CD38. EXAMPLE 7: FLUID PHASE ACTIVATION AND SAFETY BiCE polypeptide constructs were evaluated for complement activation in a fluid phase assay measuring C4a generation. The CD38-BiCEs exhibited specific complement activation in the present of CD38-expressing cells (FIG. 13). Thus, CD38 IgG BiCE constructs do not lead to complement activation in the absence of target cells. Next, a mixture of Dara-BiCE and soluble CD38 was combined in normal human serum. Surprisingly, CD38 BiCE shows no fluid phase in the presence of soluble CD38 and does not lead to complement activation (measured with C4a ELISA) further confirming that CD38 IgG BiCE constructs do not lead to complement activation in the absence of target cells. EXAMPLE 8: PHARMACOKINETIC EVALUATION OF DARA-BiCE IN A RAT MODEL. The pharmacokinetic properties of Dara-BiCE was evaluated relative to daratumumab. Daratumumab and Dara-BICE were dosed IV at 5 mg/kg and 6 mg/kg (equimolar) in SRG rats. Serum levels were detected using a generic IgG PK ELISA. Half-life, volume of distribution (Vd), clearance (CL), and area under the curve (AUC) were quantified as shown in Table 8 below. TABLE 8. Pharmacokinetic properties of Dara-Bice as compared to daratumumab. Construct Half life Vd CL AUC* days mL/kg mL/h/kg hnM dara 16.8 85.1 0.146 154519 dara-BiCE 15.2 69.6 0.132 178228 * AUC parameters calculated in nM to enable comparison between different MW All parameters are within the expected range for Dara and IgG in rat (See also, e.g., Espinoza et al, 2019; Li et al, 2024). EXAMPLE 9: EVALUATION OF DARA-BICE TREATMENT OF CANCER IN AN SRG RAT MODEL Dara-BiCE with a C1q-binding moiety that was optimized for rat C1q (Dara-BiCE surrogate); daratumumab; and palivizumab (negative control) were administered to Sprague
Dawley Rag2 -/- Il2rg -/- (SRG) rats with human B cell lymphoma (Daudi cell line) according to the schedule in FIG. 14. SRG rats have no T cells, B cells, or NK cells but have a fully active complement system. Treatment of SRG rats with Dara-BiCE surrogate resulted in a dose-dependent tumor size reduction, tumor growth control, and complete tumor clearance approximately 21 days following the 1st dose. The SRG rats treated with Dara-BiCE did not exhibit adverse reactions and maintained body weight. While some embodiments of the polypeptide constructs of present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the constructs disclosed herein. It should be understood that various alternatives to the embodiments described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the polypeptide constructs disclosed herein and that methods and structures within the scope of these claims and their equivalents be covered thereby.
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QL S F V QS SAVL Q KQR KY Y SF AE AVYYS YI T P N DH V QF NY W G YT K F NTNT S K WL A ATAVK KE S WD GG L F DVTWY L R T S VGGN L DT V DGN S I T Y YSG YN G L K S P S : Y ni S TRQ GY L KVAG F A HS GF CDS VV L a S G S I E KK S S A S R GA R R Q L S L VE G h V c S G S VA E L TG GD R KA NF R S S AA TQ th Q g S GK T R L TGF V S S A) 9 DE V GT S H T i A S F GP YS G C T WDD: G S P I GG S E V L b R a C R Q S A P GF S N L S E GA YAO N CGS T Q YK S E C I F N YG R S A S AYC D I P TL NA mu L T G T P L T L TN TSY I RR AKQQ E G SY mATP u t C S DF S C S GY I Q TSC V YDQK ) 1 a R r E A R WVK K L GA E S : QDYS P L H 1 : a GN NS V S S TR L R T D ( n i G P GDP AK O D P S S R ADV QP S W S E S S a h Y I A F I NE N d e L A i DQT E S f S YQ CG T S GI L G P AP V C A VV E F D VYD
AK L T t h S L DVSKD I i L d o TI AL YS KV S GQVN V g i VV P E AP AQ M = P L YL E QVAN Q T L S R P VQ 7 S P A E QH TS QAW QKS E S GE RMG 8 P QTAVL ( 1 QAA F D S N VGE Q L Q 0 3 Q TG GV TKT L C 5 7 T L QD S P GE GQTG O P R L KS I RAT E G MVG C I E P E P S C AS KP E F I Q L YE G VP V ATW H O E N D M I QTKE S R D QL T I E R P S L N VS N CL YG S C S VQI S E E P KS P : ni AQSG P HNLL AVYS a QLA YS SN VP V S C T K WD RS D F L YK AL P P T E R P L L C L L F S h L c R VL SN TVWNF EVL S Y CL F VK CS LK MI VAGS E R P AT F S S WNTS VI VGKVF G A F T S F P TS VS I KKL N R E GQ R T N S GK Q NKVT YSDYQF S P S MS L TVT QP GVE N KGL F GG F G S L KM VDL KS VD TKWT TAVQS T DY y vYNGP TGYK YD T D S RQE GL WGMS L S F V G S L I S A P V KDN KN KK F T K H P V C K L H a S SGP F L L N N KTVN e S P H HF I YS E F L E E D LK Q TT F WDS S P KWRVT S D S S TN K E YMP L F RYKPAP VDS P P PY VAD KP H S P VE E TA GGA F VV C E PQP TH ni AY a AAVP P GKE TE SKG L T VP P D H L TN h C Y c S T C L GKF D L GF P F S TH L N F VGR YN S KH AV TC L P Y P N M ) ASYGV E L TKH V 7C S C v SDV L S T HVYYL H S T L VNA y R GYVL VV a W L N S 8 0 LAL AL TVVQNE H e L S G S V AS P A GN S C P WT E C 3RYP A SKDS P E E GNDYP S OL Y L T YDVVR P QM H GGTGS I F QVQ F RS T D RG GL CVVPGV b GS I DKN YGKH QGV OGNSASGSVRQ S a P E T m W T L VL P N N ) G P TS SKCYGNSC u QA V S A S RAS Q L E V S 1 M ( PDCS QP T TKE S F mu L VL S V T E TG P P DT E L R E G: Q t P Q GYK O VSYS L E V SA V VVE PNK W EN ) a GWS S VL AE V r GE NV a P S P GL C H S QW E Q L I VS W N GGA P AR PKTYS RQI VG0 Q 1 D S GMT VE S KQ L L P S P S F P P VVGV DVK AA I R S D I GS T A b a GVD L F DS E T A SWE P TVI E K I F HKMR E I DQ: O SW N = E TYVC 1 L L G P Y L G TTVR YKDKQ mE EAVVT L P P P R D QDVH VT S I S P DE a VS ( t r V LR a LGL S P G SNS T KA DT P Y F S K I 9 QATGKVTVS T YT z QKSGT P KKLGD Q MVQN VS KCN K F L K l eV NK LKPAAKVE C E R K W L S D T Y E G K G F QG P M T A H K N K V T S ( 5 5 1 2 5 1 7 - - 3 e 1 5 m 9 2 1 a M N M C C
GAL P E QET AYAST P LDE TV QD GAQQA QE DVS P S P E LKS V QS S P E S KD E E P T YR L P Q L QE S T TK W SGF I SG QAS P T E V VS I F N GQ YQ H T P E T AVS S T W S G F VS YL P QV VS I L TGG NS L E AE D A YTVKH GAAAC I S N T VN KL TS P T A V T DY N S G AAPV RVV G I TAS SQ S S S K NK C KKI K N DS GS S G EWH n VQK i DG a GS PAY NQ GKS CS KVE h S I CS LWR S F TKY) c TRGAD 3 t h S I GSVAH TV S : n D i VP GEA 1 : g RF L TKaRVG F R P K S O i l AR 0 TE K TVW hQG YL N 8 QP I AQ) Ct GS V S F T 0 G GGE 6 h P P g i TRGNL D G T I 3 GGP P 2 OP S GYT : S Q S S LN L S S Q AP E R VRF VF L D S O L DS L L S OSNF S L N b a A S SC PRD S ( VN VYC M SDYI LYS D I ma P YY t T E = P S SCS Qi QI L S V QS S D G 8 PG 4 QYDVA LAE g a T L L KCA R 2 T I Y YT K S N 1 LVS TYS ( z e V MS P QA TYG DSD F K QS ME K C I VSAK DV P CKN S Y N N C E : ni P S NF S S VF a A Q PP P h c Q L W RYV L TS C V I L V Y F E VTLY A VVK S L K Q Q L QGRYCVP F G R F YTSGHGV KF L S S CDQP TH E H L TN T L TKH VTV L TT SGCVF P AGAV PAL H H SVYYL NTVQ PDKQS S WMQASWA S TVT LVNA WKGP TGVYNGL S ANGCSDRT YKD QNE S S E GGYE K D L MKWLAYGLDVV S P E H MNQP F L LWKTS S VDYRYMGGCVVE P M G YDG RWYS L NGMDK DNDL F NS S SVVRQV DS S E F N E L Q NDYS T T S SKCRPGS DS I H P PKL EVT n i NRAGNRK QP T QNC DF T F GK F VVA TPVW E DR S P P Y a h F TS I S S G SA PGCS L SVE VVYTGS S SKE F T Y LVC P P QP TH c RV QDYP A R E P NAWV F QF CVP D H P TN y GF W SYA PKTYK E Ny GGGGS C E L LKH v S R R VYVL F DR SVGv SKS L S T TYL a AGS P L I AP T SQI AQa eAV SAL H SVYNA e K GN TF HVI E TI Q) HA S CDDA TS H TVT LVNE h A QE H 0 CV SWGS GGVDVSVK G TME KD L R E I SW5 2 SVH YKDVP P 8 SDP SWE P SNTP P P R : LYF GL DVS EQM 0 LAP E E AGTSDK Y SO R L H L S G SGS S T GT S C SVV KV CVR R PGV S 3 R OLYN QN E V SC R S HDL L RKL P TA QGL S T SAKK HP KP F K ALGD N AKVD GKR S QP TYGE S OGTAVKNAG SNK VT I GGAK PGCS L E SVVV E T SK F GGG G AWV MAG Q S P M QS SNVP NP NK F HN L CLQ KE QNY V YP ARP N E N d QGC LWS A P KTYKVG e i VAYGA QL YV TW S C I L F V S EVT L S S Y ( I VL F TDR S SQI TA I Q) f Q2 i d LGI Y I G GR VL TVV TYVVKS K TSGCVL G A b aGD S T P F HVI E D 1 VK o GL AG L V P DK F ME K SW: GATSWNT P QG YQF G S P S mG DV a SVE S t P G T S R E NL P I P P YR SO mG N = TVD EGHK SG QE T P GGV YEN KGL S i EWA g a L z L E RGTS T PDK TA L LKL P F K E F SGMNGF L L G D 7 4 V E KGGD YS L WK GT D S L S AKKKL D KV I 2 LR E LGSYRWDS S E P N F NM E DK eQGS T P A Q 1 S S SY L L MV EKNS G MAG S SH NK AVT Q N VP N P K H NL CLE MVT KS ( C QGNS P MV S T F I T TDK F F F V P L VA TP K CV EWE DRVQ S P P T Y 96 8 2 4 1 2 - 1 7 - 6 7 2 4 1 2 1 M M C C
AY STTGY AQVN A V QDA P G QQ W L QL QGA APQ P A E LKA K VG S VA R F YTI T NNK S P S C K QD E S E E P T S TKASA AG TDR WMQDG AA ANGGSQDS AY S NGKS QAS TE CYS MK CSL R YQ P WT P T EV TS W LYA D VDYS I GVW STAH S S G F LV GS GRT SA) n DN i D ND R Y F L V TK n VS I TT L VAE SGA 8 C 2 a NRAA TR E K TVW i a Y h KL SKHGG RY: h c F TS I S SQP I GAQ E ) c t NTP V T h GA PVG P O WY t h RV G PGGG P P 0 TAS 3 QQS I I A N g i GF R W RAS P G F YT : g I A NNK S DCV SAT D I L S AGS VRV F L SO i l DG 0 S PAS YGADQ 0 AKP S WVNF YD SL N Y 8 GNQ GKSGVE P E 8 0 CV SGSN DS I L S D I 0 S 3 CS LWRGA S E K L S ( 3 S LDP P PGS DCSQO S I GSVAH S E RNS S O RAP NQY I YVA LAER RF T L V TKV E NV R O L SY H E TVS S TYS (O A TR E KVWLQ QGMT M GTDL AE I VCAKSM QP I TAQ E VP QV L Q d GGGD V PYKN N C N E d e GGG i GG P P QA SQTT e i AG QGCSAY DAS T f P i AS P GYT L GR VG f i VAYG GQAQQP A d o V SRF F SGF TQ d NV F D S NG o LGI Y I GG P E L EKS V I L GWK G W M GL A TGKD E E P T T M V = SN PDYLYS G S G L AH = GADS GQAS S T K E 0 PGS SCS N AGNDD = TVF E Q SGYQ P 5 QY P TV2 1 T I DV LAGYRY 1 E 5 V EKG T LVYS TYG WG F E VT S GA S S I E Y 2 LR E L GS SS I LGGM E I VSAK CKNSDTG 1 QTS NSVT T VAE C DV P Y N NC E R F F RT S M V C QG P MV TY KL T S PK VH T AQ L SWNF VSTS AQ LSWNF VSTS QYV TS C I L F E VLYK QYV TS C I L F EVL YK RL VVYVVK GCS L F G RLVVYVVK GCS L F G VT L TT S V WNTV PDKQF S P S VT L TT S V P DK F P S HK P Q TGVYNGL WNTVP Q T Q GVYNS L MSG QE GGYE K D S L HK SGE GGYE G K D S L GN YDGP F L S L K LWGTS S MNQ GP F L L WK GTS S S RWYS E N SYDS P P F NMD L E L K GD S L E WYS E N NMD LK VQ Y S R SYDS P F L E E VQ S F I DKVAK PVWR P T Y S I DKP VAK VWR P T Y TT F F F V T GRF L C E DS P TH F T F F V T P CEDS P TH GYCVP P QP P TN T F RF L CVP PQP P TN HGVS P D E H L LKH GGY HGVP D E H L L KH n i SKF L S C T HVTYL n SKF L S S C T HVTYL a A AV PAL H S TYNA E i a A AV P AL H S TYNA h c CS DWA S TVLVN S Q H h CDWA STVLVNE H y S v LART YYGY G L KDVP E P c SART Y GDVS EQM V y v LYYG GLKDVQP E P GDVS E QM a e R L F MS S C H S T N T S SVVR KVV R PGS a RF MS SC SVVRGV S QNC e L S T N T SKVV R P NC 0 GN R S QP C TYGS S H GN R S QP C T Q YGS S 8 G S GAK S L E VTKE F 0 8 G SAK L E VTKE F 0 3 P QDCO V SYSV AVE SAWV GCSV N 0 P D S VE SAWV N YP P RP TNK E Y G 3 QS P A PRPNK E GR L Y I VA L F K TDRYSVO GN S A TP F HVSQI I E T A I Q) O VYY VAF KT RYSV Q) DQ7 2 R L I L TD O GN S A TP F HVSQI I E T A I DQ7 2 M GVDVVKME K E SW: M GVDVVKME K E SW: = GS E S G T L R P I P R 6 SO G E S G T L R P I P R S O EWAP SN 4 S T K P Y VE RGT PDTAP F K N = S EWAP SN G D 6 4 ERGTS T P DK P Y TAF K N G D 2 L L LKL KKKL D K I 2 V L L L KLKKKP L D K I 1 QGS TAHP AAVV TQ 1 QGS TAHPAAVV TQM VKNS G SNKNKL L E M VKNS G SNKNKL L E C QG P MA S N VP P H N C KS ( C QG P MA S N VP P H N C K S ( 0 5 1 2 5 1 2 - 1 6 -4 6 2 4 1 2 1M M C C
QYSNVP H KV S NF VN SL CL K NTS F AKDS VT V TW S C I L F EV V T L S G YK PVLQ E K T WKSVV T Y TV K SGCS VL K G D Q Q E E T VV QS S P DK F P QTDS P MKTV DGP Q E TGVYQF S ENS L YQS P E S S W Q GGY KGS WL P Q L YA D TGP F L S L L WK GT D S L S VS TF I S NG Q TL TWY DS E P N F N L E DK L I RTF VG S H F AY S P KLDL Q L S T T DK AVWRVT NTP QV YK GL S V L V TP CE PDS P P PY Y I YAL AE G CVP QP TH DDANC S Q F N VP D H TN EKV A A KKS GG L S S C E T H L L TKH S AG S TD VY V CQ AYA YAL S H SVYYL K RK VTVNA GK K S L YDT Y T KDL KG VQNE C S I WH H T I GEQK VTGGL RGGDV VS P E SD VE PM TS L VF KVE GAS TS S C SVVR P Q GV RR S TKY AD) : T n i G P DC GYS QKP C TRQ S DP G Y NC VGEA4 3 YK S L E VTG KS E S : G L GGR P K S : a hKP YVSV AVE P SA F WV 3 1 S T F TYL O
cAt I A P PK N VE F T N h V g i E T GS A KT K EN ) 3 S L Y L DL F TDRY QS I VG2 MF S PN NT D I l AI E PHVS I E T A I Q 3 : CS T TL SQ b a G SWS F VVKME K E DQO n S i SVS WL S L E QEA L L S G T L R P I S PW N a SGSYCS S ( m i x VGS P GSNS T K P AYR S D I h Q TI QVYC u t L QS L KT L P D K T P F K C t L ML QV S T S E aQ K K VG PHT SA P L GD Q E h g QGCADG s I Q R M AG SH NK PAAK N K V S i I P YT KR V T ( L D A Y G S N QYSNVP HNL L G KLP KLKV KV S WNF L V S CK P SM KTAK K PAAVTQ VT V TS C I F EV V T L S Y QF S GH NKNKL L E WKVVYVGK CS LK R F AS S VP PHN CK S S ( QS S S TT S P DKVF GP LVSN NF V S VT LYK MKTVP QGVYQF S S WQ SVW S CL E KS L G WDGE T GGYENGL S H K T L V I YF V VGCVF P YAQP D T LGF L L WKK YS T D L I S T TT SDKQF S S L S L NGL S S GNTVP Q P N GVY GS TTWDS E P F N L E D LK Y SDGE T YE K TD L F TAY K D Q KQ GG K S S DKP A VT TT P L LWG K YK GGL S V L V TP VEWR S PY L I GF S L N NM E D L Q S Q F NCVCPD QP PH S F S VP P D H P T TN G L WY F DS E F L EVT KS P PKWR P Y AKS G L S C E L L TKH S R S D F VVA PVDS P H KQG YAS T HVYYL VMY L TCE P QP P T TN CSAYAL S H S L YDT Y TVTVNA T KDL C F F CVP VQNE TAG TGVS P D H LKH C E L TYL KGGGLDVS P VT E E PH I SATL S T HV TYNA E : SDRG SGCVVRQM LNVA AL H S V VLQNH n i TGATS S SVVP GV DCS QKC S SY DKTS E TRQNC : QTGGY TD L K VP P VS EQM a h G c P KGY PYK S L P VE YGS S 3 S VV E T P SKGGDVVRGV S yAYVS P AKP SKE F 1 QGAS S CVVPNC NAWV 3 GCT S S S KCRQS S v VI TA S A PKTYK EN ) MMRY QP TYGE F a e E DL F hAG I TDRQS I VG1 L WY I K S L E VTKWV b a G SWE S P F HVS K I E T E K A I Q 3 Q: CG O n i P I SVAE SAE N GVA TP A P KP NKVG mQEAVV i L L S TM P GNL R E D P I W a S GDA P S P N hQL L F TKTYS RQI TA I Q) Q3 3 x V u t L GS S S T K QSGT L P DTAP YR F S D C K I y K D LW P DS I E KD E GF HV W: ME E S O aQ L K KKK VG PHT SAHPAL GD Q vQ AK E a e L QVVK GL S S PGT L R TP I P P YR S N s I Q R M AG S NK P N K VV T S ( HV E K N GS TN S DK TA P F K G DD I b a mi x 3 u t 1 3 a s M I C
e fi e l f - i e f l f e f e f e f e f e f e f e f e f e f e f e f e f e f e f l - i f l l - i f l l - i f l il il il il il il il il il il il il l -fl -fl -f -f -f -f -f -f -f -f -f -f -f a a a a a a l a l l l l l l l l l l h h h h h h h a h a h a a a a a a a a e e e e e e e e e h h h h h h h h
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g n i d n i b g n R i y d c n e i s b a e F R ) ) e r s c a n n e i y s t i r c s t c c d a c F e f e f d n l i e d r e f e f e o d l t s w o l C F C ei e e e e e e f o o , F f l f f f e e e e 0 f m d d - i f l l - i f l l - i f l f l - i f l f - i f l f - i f l f - i f l f - i f l f - i f l f - i f l-f 0 2 0 6 l a g - n g i n d i e d z i e z i a h a h a l h a l h a l h a l h a l h a l h a l l y h a h a h l n , y , l y l y l n i n i m i m n i e e e e e e e e e e e o n o n o n o b b q i n i
v o v r o v p r o v v v v v v v v R r o r o r o r o r o r o r o o y q 1 q 1 q 1 1 q C 1 m C ( m g ( g m p p p p p p p p r p r p c F C C C d i / m r i / m e r i / m r i / m r i / m m m m m m g g g g e d n i n i s e s d d r i / r i / r i / r i / r i / r i / r n i n n ntl etl et d i d i d i d a e a e n i n b i b A l et A l et A l et A l et A l et et et et n n n n r c r c A A l A l A l A l A i B i B i B i B n I n I o N o N V8 V 7 8 V 8 1 3 7 7 Gg A 3 A 3 A I V , 1 V / R / / d 1 8 7 V V e t 3 7 0 1 1 1 1 a l y Q 3 / A 3 T 3 3 R 7 D / / Q Q s o 0 5 V V 8 V 8 V 8 D / 6 R V V / R c y l G A 3 1 1 T 3 1 7 / N 3 3 7 7 8 7 0 8 7 8 7 7 0 g a 9 2 5 A 3 3 2 6 D / Q / A A N 3 T 3 A 3 A 3 T n i 3 P 2 6 Q / V / D / V / D / D / V / Q / D S , D, 8 7 R 2 0 7 1 3 0 1 5 1 5 6 1 7 5 R 6 R 6 3 3 8 2 1 3 8 2 8 2 1 3 0 3 8 2 8 3 8 3 3 A 3 5 3 E 5 3 N / T / 3 T/ Q / H Q H H Q T / H Q Q E , 2 L 2 LD N D D / D / D / D / D / Q D / A , E , E W 3 , , 6 5 5 5 5 D 2 0 0 6 / A F 2 8 6 2 5 2 8 6 6 6 6 7 2 5 2 5 5 5 0 8 6 1 4 4 2 5 2 3 2 3 2 3 2 3 G 3 3 T H T H T 2 T 2 T 2 T 3 T H 2 T K K K K g I 2 L 2 L
e r a C s e C g oit D a h a r A p o b d n C r c g 2 n 3 a C a C D A m id D ni C/ D n b a C d 6 , n e a h w q 1 1 e f C t C D il p r C -f o l D e c / r e t a C h d e x dn t a e b d e e n v ( o P g , n g a i n d i g r p C D d . n d n i m I A e s C i n i d n . e d a C b b i b fil n a e r c D R R n -f l C n i A n
yc y F c y F c F a h D C o t i e de d d d d e v e v e v n a e v u s a d e r o o o g s c r r r o l n p m p p g n i r I m I m I n i d p l e i t c n i m I cr n a n b o a t h n c c i e f i n y e c f F f n e g i s L8 2 4 M/ L L 6 6 9 E 9 3 2 3 P / 3 3 L 6 P / L I 9 I 3 5 5 5 T 4 P 0 / 3 3 2 L 5 V R / / 3 R S 1 F 5 L 3 0 1 R 0 3 8 6 / 3 A Y 6 / P L Q / 2 8 A H E 3 E 2 2 6 3 7 2 2 9 4 2 6 G 3 3 2 M 2 / I R G / S V , / , 1 D R V A 3 2 9 L 3 3 1 1 1 3 6 3 A 2 4 S 2 F 3 Q 2 A 2 G
Table C: some preferred but non-limiting examples of non-naturally occurring Fc sequences (illustrated using the Fc portion of the anti-CD20 antibody 11B8 as a reference). Sequence of Fc region of 11B8 (for reference only): CDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 14) 11B8 Fc [K320E,Q386R] CDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYECKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGRPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 15) CDKTHTCPPCPAPELLGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 16) 11B8 Fc [K326W,E333S] CDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNWALPAPISKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 17) L234A/L235A/P329G (LALAPG) CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 18) >Heavy chain sequence (11B8 Hc K320E,Q386R) MGWSCIILFLVATATGVHSEVQLVQSGGGLVHPGGSLRLSCTGSGFTFSYHAMHWV RQAPGKGLEWVSIIGTGGVTYYADSVKGRFTISRDNVKNSLYLQMNSLRAEDMAVYYCAR DYYGAGSFYDGLYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK RVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYECKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGRPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 19) >Light chain 11B8 Lc-5GS-hNb78(M33A,T102A) MGWSCIILFLVATATGVHSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQK PGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSDWPLTFGGGT KVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSQVQLVES GGGSVQPGGSLRLSCTASGWTFRDSAYNLGWFRQAPGQEREAVAAISWRGGSTYYADSVK GRFTISRDNAKNTVTLQMNNLKPEDTAIYYCAADASARAALYSTGYEYDHWGQGTQVTVS S (SEQ ID NO: 20)