Engineered complement engaging polypeptides

Abstract

Disclosed herein are C1q complement factor-engaging polypeptides with optimized C1q-binding affinity, resulting in improved complement-mediated immunological responses against target antigens. Also disclosed herein are bispecific constructs that comprises: (a) a C1q complement factor-engaging domains described herein and (b) an antigen binding moiety that binds to a target protein implicated in a disease or condition. Also disclosed herein is a method treating a disease or condition in a subject in need thereof comprising administering a bispecific construct described herein.

Description

ENGINEERED COMPLEMENT ENGAGING POLYPEPTIDES CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to European Patent Application No. 24173650.3, filed 1 May 2024, European Patent Application No. 24173646.1, 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. BACKGROUND

[0002] The immune system is involved in the pathogenesis of several diseases, including but not limited to cancer, autoimmune diseases, obesity, neurodegenerative diseases, and cardiovascular diseases. At the same time, the immune system can be harnessed to treat various diseases or can be targeted to reverse disease pathogenesis. The ability to harness the immune system for therapeutic purposes has been tenuous in the field of medicine as there is a delicate balancing act of maintaining both pro-inflammatory function and anti-inflammatory function of the immune system while still targeting the root cause of the disease.

[0003] 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).

[0004] The international application WO2019 / 238674 also describes a number of specific Nanobodies against C1q, including the Nanbodies 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 (see SEQ ID NO: 3) and IF78 is also referred to herein as Nb78 (see SEQ IDNO:2)]. WO2019 / 238674 also describes that the Nanobodies described in this application may be humanized. For Nb75, reference is also made to Pedersen et al., Front. Immunol.11:1504 (2020).

[0005] 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).

[0006] 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 or 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 or 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.

[0007] 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”. SUMMARY

[0008] Disclosed herein is a C1q complement factor-engaging polypeptide that comprises one or more modifications relative to a corresponding wild-type C1q complement factor-engaging polypeptide, said one or more modifications resulting in reduced binding affinity to a C1q complement factor as compared to a binding affinity of the corresponding wild-type C1q complement factor-engaging polypeptide to the C1q complement factor, wherein a cancer-antigen targeting bispecific construct comprising the C1q complement factor-engaging polypeptide exhibits an increased C1q-mediated immunological response to a cancer cell expressing the cancer antigen, as compared to a corresponding bispecific construct comprising the wild-type C1q complement factor-engaging polypeptide. In some embodiments, the C1q complement factor- engaging polypeptide is an engineered C1q complement factor-engaging polypeptide. In some embodiments, the increased C1q-mediated immunological response is determined by an in vitro assay that comprises: (a) linking the C1q complement factor-engaging polypeptide to an antigen binding moiety that binds the cancer antigen expressed by the cancer cell, thereby generating a bispecific construct that comprises the C1q complement factor-engaging polypeptide; (b) contacting the bispecific construct with the cancer cell in the presence of the C1q complement factor and determining an EC50 against the cancer cell; and (c) comparing the EC50 of the bispecific construct that comprises the C1q complement factor-engaging polypeptide against the cancer cell to an EC50 of a control bispecific construct that comprises the corresponding wild-type C1q complement factor-engaging polypeptide linked to the antigen binding moiety that binds the cancer antigen. In some embodiments, the one or more modifications are present in a C1q binding region of the C1q complement factor-engaging polypeptide. In some embodiments, the C1q complement factor-engaging polypeptide has a binding affinity to the C1q complement factor of from about 10 nanoMolar (nM) to about 2 microMolar (μM), as determined by biolayer interferometry. In some embodiments, the complement factor-engaging polypeptide binds to C1q complement factor with a binding affinity of about 0.1 μM to about 2 μM, as determined by biolayer interferometry. In some embodiments, the complement factor-engaging polypeptide bindsto 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. In some embodiments, the C1q complement factor-engaging polypeptide comprises a single domain antibody. The aforementioned ranges are also referred to herein as the “ranges specified herein”; it being understood that this wording, when used herein, generally also include preference(s) for the subranges as mentioned herein. In some embodiments, the C1q complement factor-engaging polypeptide comprises a chain constant region (CH), a heavy chain variable region (VH), a light chain constant region (CL), a light chain variable region (VL), a VHH domain, or a VNAR domain, a DARPin polypeptide, or a KNOB domain peptide. In some embodiments, the one or more modifications comprises one or more amino acid substitutions relative to the corresponding wild-type C1q complement factor-engaging polypeptide. In some embodiments, the corresponding wild-type C1q complement factor-engaging polypeptide has the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3. In some embodiments, the one or more amino acid substitutions are each at a position corresponding to a position in SEQ ID NO: 2 ranging from residue 32 to residue 114. In some embodiments, the one or more amino acid substitutions are each at a position corresponding to a position in SEQ ID NO: 2 selected from the group consisting of: residue 32, residue 53, residue 59, residue 74, residue 101, residue 102, residue 103, residue 104, residue 105, residue 106, residue 113, residue 114, and any combination thereof. In some embodiments, the one or more amino acid substitutions are each at a position corresponding to a position in SEQ ID NO: 2 selected from the group consisting of: residue 101, residue 102, residue 113, residue 114, and any combination thereof. In some embodiments, the one or more amino acid substitutions are selected from the group consisting of: D101A, T102A, Y113A, E114A, T102A / Y113A, T102A / E114A, and any combination thereof, as compared to the corresponding wild-type C1q complement factor-engaging polypeptide having the amino acid sequence of SEQ ID NO: 2. In some embodiments, the one or more amino acid substitutions comprise: D101A, T102A, Y113A, E114A, T102A / Y113A, T102A / E114A, or any combination thereof, as compared to the corresponding wild-type C1q complement factor-engaging polypeptide having the amino acid sequence of SEQ ID NO: 2. In some embodiments, the one or more amino acid substitutions comprises the Y113A amino acid substitution, relative to the corresponding wild-type C1q complement factor-engaging polypeptide having the amino acid sequence of SEQ ID NO: 2. In some embodiments, the C1q complement factor-engaging polypeptide comprises an amino acid sequence with about 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 4. In some embodiments, the one or more amino acid substitutions comprises the E114A amino acid substitution, relative to the corresponding wild-type C1q complement factor-engagingpolypeptide having the amino acid sequence of SEQ ID NO: 2. In some embodiments, the C1q complement factor-engaging polypeptide comprises an amino acid sequence with about 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 5. In some embodiments, the one or more amino acid substitutions comprises the D101A amino acid substitution, relative to the corresponding wild-type C1q complement factor-engaging polypeptide having the amino acid sequence of SEQ ID NO: 2. In some embodiments, the C1q complement factor-engaging polypeptide comprises an amino acid sequence with about 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 6. In some embodiments, the one or more amino acid substitutions comprises the T102A / Y113A amino acid substitutions, relative to the corresponding wild-type C1q complement factor-engaging polypeptide having the amino acid sequence of SEQ ID NO: 2. In some embodiments, the C1q complement factor-engaging polypeptide comprises an amino acid sequence with about 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 7. In some embodiments, the one or more amino acid substitutions comprises the T102A / E114A amino acid substitutions, relative to the corresponding wild-type C1q complement factor-engaging polypeptide having the amino acid sequence of SEQ ID NO: 2. In some embodiments, the C1q complement factor-engaging polypeptide comprises an amino acid sequence with about 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 8. In some embodiments, the C1q complement factor-engaging polypeptide comprises an amino acid sequence of any one of SEQ ID NO: 4 – SEQ ID NO: 42. In some embodiments, the complement factor-engaging polypeptide modulates the activity of a complement system by directly or indirectly activating the complement system. In some embodiments, the complement system is human complement system. In some embodiments, the complement factor-engaging polypeptide directly binds a C1q complement factor. In some embodiments, the complement factor-engaging polypeptide indirectly binds a C1q complement factor. In some embodiments, the C1q complement factor is human C1q complement factor.

[0009] Without intending to limit the scope of the invention in any way, in the present specification, the invention will be described in more detail with reference to one of its preferred embodiments, in which the C1q complement factor-engaging polypeptide (also referred to herein as the “C1q binder”) is a single domain antibody (also known as an “immunoglobulin single variable domain” or “ISVD”), such as a single domain antibody that essentially consists of or comprises a suitable heavy chain variable domain. Examples of the kind of single domain antibodies that can be used in the invention will be clear to the skilled person based on the disclosure herein and the publications relating to single domain antibodies cited herein. According to one specifically preferred but non-limiting embodiment, the single domain antibody willessentially consists or comprises a Nanobody (including VHHs obtained from a species of camelid as well as for example synthetic or semi-synthetic VH sequences that comprise the co-called “hallmark residues” that are characteristic of VHHs and nanobodies, for which reference is also made to publications relating to single domain antibodies cited herein).

[0010] When a C1q binder with reduced affinity for C1q (also generally referred to herein as a “C1q binder of the invention”) is an ISVD, as described in more detail herein, said C1q binder of the invention comprises one or more mutations in its amino acid sequence that reduce the affinity for C1q (i.e. compared to the affinity for C1q of the ISVD from which it is derived) such that the C1q binder of the invention has reduced affinity compared to the ISVD from which it is derived (and preferably a reduced affinity that is within the ranges cited herein). When such C1q binder is an ISVD, said one or more mutations that reduce the affinity for C1q may in particular be in the CDRs, such as in CDR1, CDR2 and / or CDR3, for example in CDR2 and / or CDR3. According to one specific but non-limiting aspect, said mutations comprise at least one mutation in CDR3 (such as one or two mutations in CDR3). Also, the total number of such mutations in the ISVD will generally be 5 or less (such as 5, 4, 3, 2, or 1), in particular 3 or less (i.e. 3, 2 or 1) and as can be seen from the further disclosure herein, a single mutation (or more generally, “amino acid change” as defined herein) in one of the CDRs (and in particular in CDR2 or CDR3, such as in CDR3) may be sufficient to reduce the affinity for C1q (preferably to within the ranges cited herein). As also further described herein, C1q binder of the invention that comprise one or more of these mutations may further comprise one or more other suitable mutations, such as, for example and without limitation, one or more humanizing substitutions. Alternatively, said mutations can be introduced into (the sequence of) an ISVD which ISVD is a humanized variant of a naturally occurring ISVD (as further described herein). Some specific but non-limiting examples of suitable mutations (or combination of mutations) will be clear to the skilled person based on the disclosure herein and / or can easily be determined by the skilled person based on the disclosure herein, and may depend on the specific C1q binder that is used as the starting sequence. For example and without limitation, as illustrated in the Experimental Part, when the C1 binder is a variant of Nb78, it may be advantageous to use a suitable combination of an M33A mutation in CDR1 together with one or two suitable mutations in CDR3 (for example M33A in combination with at least T102A or S103, as further described herein). When the C1 binder is a variant of Nb75, it may be advantageous to at least include a T53G mutation.

[0011] As further described herein, further aspects of the invention relate to methods for generating the C1q binders described herein, which methods comprise (at least one step of) suitably introducing (as further described herein) into the sequence of a naturally occurring (asdescribed herein) antibody sequence against C1q, such as into the sequence of a naturally occurring VHH sequence against C1q, one or more (suitable) mutations (as described herein) that reduce the affinity of said naturally occurring sequence for C1q; and may further comprise, for example and without limitation, humanizing the sequences thus obtained (or alternatively, as further described herein, in order to obtain a humanized C1q binder as described herein, a naturally occurring antibody sequence against C1q may first be (suitably) humanized, after which, one or more (suitable) mutations (as described herein) that reduce the affinity of said naturally occurring sequence and / or of said humanized sequence for C1q may be introduced). Further aspects of the invention relate to the C1q binders thus obtained.

[0012] Such mutation(s) may be any suitable mutation(s) (which can be determined by the skilled person based on the further disclosure herein, optionally after a limited degree of trial and error). According to one specific but non-limiting aspect, such mutation comprises mutating an amino acid residue that is not alanine into alanine (or into a similar nonpolar uncharged amino acid, such as for example valine, leucine, isoleucine or proline).

[0013] As will be clear to the skilled person based on the disclosure herein, when the one or more mutation(s) that reduce the affinity for C1q involve mutating an amino acid residue that is not alanine into alanine, C1q binders of the invention may for example easily and conveniently be identified (i.e. starting from an ISVD that can bind to C1q) by performing so-called “alanine scanning” of the CDRs (and in particular CDR2 and CDR3, such as CDR3) of the starting ISVD and identifying ISVDs with reduces the affinity for C1q compared to the starting sequence. Thus, in one embodiment, a C1q binder of the invention may be provided by the steps of: (i) providing the sequence of a C1q binder that can bind to C1q (and preferably with an affinity that is better than the values that fall within the ranges cited herein); (ii) providing a collection or library of mutants of said (starting) sequence, in which each mutant contains, within its CDRs (and in particular CDR2 and CDR3, such as in CDR3), a mutation of one amino acid residue that is not alanine into alanine; and (iii) testing said mutants for affinity for C1q (and in particular, determining the affinity of each of said mutants for C1q and comparing it to the affinity of the starting sequence); and (iv) identifying (and optionally isolating and / or producing) mutants that have reduced the affinity for C1q compared to the starting sequence (and preferably an affinity for C1q that is within the ranges cited herein). It will also be clear to the skilled person that, for this purposes, other techniques known per se that are similar to alanine scanning may be used. In one specific but non-limiting aspect, the starting sequence may be Nb75 (SEQ ID NO: 3) or a humanized version of Nb75 (such as for example SEQ ID NO: 27) or Nb78 (SEQ ID NO: 2) or a humanized version of Nb75 (such as for example SEQ ID NO: 10). For example and withoutlimitation, Table H below shows the result of alanine scanning of the humanized Nb78 variant of SEQ ID NO: 10 (with an additional M33A) mutation. As can be seen, a number of variants of the SEQ ID NO: 10 [M33A] sequence with an affinity for C1q within the range(s) specified herein could be generated and identified. It is envisaged that the skilled person could use essentially the same or essentially similar techniques to generate and identify other C1q binders having an affinity for C1q within the range(s) specified herein, for example and without limitation taking the sequence of one of the C1q binders disclosed herein as a starting point. It should also be noted that in further aspects, the invention relates to the C1q binders that are disclosed herein (i.e. by means of a specific amino acid sequence given in the present specification) and that have an affinity for C1q within the range(s) specified herein. The invention in other aspects also relates to variants of these C1q binders that have an affinity for C1q within the range(s) specified herein, which variants can in particular be as further described herein and / or which variants have been (and / or can be) generated by one of the methods and techniques described herein. For example and without limitation, such variants can be generated in this manner taking the sequence of Nb75 as a starting point, taking a humanized variant of Nb75 as a starting point, taking the sequence of Nb78 as a starting point, taking a humanized variant of Nb78 as a starting point, and / or taking the sequence of any of the other C1q binders listed in Table A as a starting point; and such variants and constructs comprising the same form further aspects of the invention.

[0014] Thus, in one specific but non-limiting aspect, a C1q binder as used herein is a variant of Nb75 (SEQ ID NO: 3) or a variant of a humanized version of Nb75 (such as for example SEQ ID NO: 27), in which in at least one of the CDRs (and in particular in CDR2 or in CDR3, such as in CDR3), one amino acid residue that is not alanine has been changed into alanine (or into a similar nonpolar uncharged amino acid, such as for example valine, leucine, isoleucine or proline, but preferably into alanine), such that said variant of Nb75 has a reduced affinity for C1q compared to Nb75 (and preferably an affinity for C1q that is within the ranges cited herein). In particular, such a variant of Nb75 may comprise a CDR2 that comprises at least one alanine residue at a position where, in CDR2 of Nb75, an alanine residue is not present; and / or may comprise a CDR3 that comprises at least one alanine residue at a position where, in CDR3 of Nb75 an alanine residue is not present (in which the total number of such alanine residues not present in CDR2 and / or CDR3 of Nb75 is five or less, such as 3, 2 or 1, and in particular only 2 or 1). According to one specific but non-limiting aspect of the invention, such a variant of Nb75 may comprise a CDR3 that comprises at least one alanine residue at a position where, in CDR3 of Nb75 an alanine residue is not present (in which the total number of such alanine residues not present in CDR3 of Nb75 is 3, 2 or 1, and in particular only 2 or 1). Again, all these variants of Nb75 should be such that theyhave reduced affinity for C1q (and preferably an affinity for C1q that is within the ranges cited herein). Some preferred mutations (and preferred combinations) for reducing the affinity for C1q will become clear to the skilled person based on the disclosure herein (including Table A below). Also, as mentioned herein, such variants of Nb75 may further comprise one or more other suitable mutations, such as, for example and without limitation, one or more humanizing substitutions (with one particular example of a substitution that may be present being T53G.

[0015] The CDRs of Nb75 (according to Kabat as applied in WO2008 / 020079) are as follows: CDR1 = NDVMA (SEQ ID NO: 258) CDR2 = LITAGGGTHYADSVKG (SEQ ID NO: 259) CDR3 = DENPPGWPSRWSSAYDY (SEQ ID NO: 260) Table G lists the affinities against C1q (globular head – determined as set out in Example 1) of a number of exemplary variants of Nb75 with affinities for C1q within the ranges specified herein. Such variants (i.e. with affinities for C1q within the ranges specified herein) as well as constructs comprising one or more such C1q binders (which constructs can be as further described herein) form further aspects of the invention.

[0016] In another specific but non-limiting aspect, a C1q binder as used herein is a variant of Nb78 (SEQ ID NO: 2) or a variant of a humanized version of Nb78 (such as for example SEQ ID NO: 10), in which in at least one of the CDRs (and in particular in CDR2 or in CDR3, such as in CDR3), one amino acid residue that is not alanine has been changed into alanine (or into a similar nonpolar uncharged amino acid, such as for example valine, leucine, isoleucine or proline, but preferably into alanine), such that said variant of Nb78 has a reduced affinity for C1q compared to Nb78 (and preferably an affinity for C1q that is within the ranges cited herein). In particular, such a variant of Nb78 may comprise a CDR2 that comprises at least one alanine residue at a position where, in CDR2 of Nb78, an alanine residue is not present; and / or may comprise a CDR3 that comprises at least one alanine residue at a position where, in CDR3 of Nb78 an alanine residue is not present (in which the total number of such alanine residues not present in CDR2 and / or CDR3 of Nb78 is five or less, such as 3, 2 or 1, and in particular only 2 or 1). According to one specific but non- limiting aspect of the invention, such a variant of Nb78 may comprise a CDR3 that comprises at least one alanine residue at a position where, in CDR3 of Nb78 an alanine residue is not present (in which the total number of such alanine residues not present in CDR3 of Nb78 is 3, 2 or 1, and in particular only 2 or 1). Again, all these variants of Nb78 should be such that they have reduced affinity for C1q (and preferably an affinity for C1q that is within the ranges cited herein). Some preferred mutations (and preferred combinations) for reducing the affinity for C1q will becomeclear to the skilled person based on the disclosure herein (including Table A) and for example may be suitably chosen from (in CDR1): M33A; (in CDR2): G57A, S58A, T59A and / or from (in CDR3): D101A, T102A, S103A, R105A, L108A, Y109A, S110A, T111A, G112A, Y113A, E114A, Y115A, D116A and H117A (or a suitable combination thereof), with T102A and S103A having been found to be particularly useful.

[0017] Also, as mentioned herein, such variants of Nb78 may further comprise one or more other suitable mutations, such as, for example and without limitation, one or more humanizing substitutions (with one particular example of a substitution that may be present being M33A).

[0018] The CDRs of Nb78 (according to Kabat as applied in WO2008 / 020079) are as follows CDR1 = DSMYNMG (SEQ ID NO: 261) CDR2 = AISWRGGSTLYADSVKG (SEQ ID NO: 262) CDR3 = DTSARAALYSTGYEYDH (SEQ ID NO: 263) Table F lists the affinities against C1q (globular head – determined as set out in Example 1) of a number of exemplary variants of Nb78 with affinities for C1q within the ranges specified herein. Such variants (i.e. with affinities for C1q within the ranges specified herein) as well as constructs comprising one or more such C1q binders (which constructs can be as further described herein) form further aspects of the invention.

[0018] Also disclosed herein is a bispecific construct comprising: (a) a C1q complement factor- engaging domain; and (b) an antigen-binding moiety that binds to a target antigen; wherein the C1q complement factor-engaging domain has a lower affinity for C1q complement factor, as compared to an affinity of the antigen-binding moiety to the target antigen, as measured by biolayer interferometry, and wherein the bispecific construct facilitates increased complement dependent cytotoxicity (CDC) via the C1q complement factor against a cell expressing the target antigen, as compared to an CDC level facilitated by otherwise comparable bispecific construct that comprises a C1q complement with a binding affinity for the C1q complement factor that is substantially equal to or greater than the binding affinity of the antigen-binding moiety to the target antigen. In some embodiments, the complement factor-engaging domain comprises a single domain antibody. In some embodiments, the complement factor-engaging domain comprises a chain constant region (CH), a heavy chain variable region (VH), a light chain constant region (CL), a light chain variable region (VL), a VHH domain, or a VNARdomain, a DARPin polypeptide, or a KNOB domain peptide. In some embodiments, the complement factor-engaging domain comprises about 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 1 - SEQ ID NO: 42. In some embodiments, the complement factor-engaging domain comprises about 90%,95%, 97%, 99% or 100% sequence identity to any one of SEQ ID NO: 4 - SEQ ID NO: 42. In some embodiments, the complement factor-engaging domain comprises about 90%, 95%, 97%, or 99% sequence identity to any one of SEQ ID NO: 1 - SEQ ID NO: 3. In some embodiments, the bispecific construct comprises a C1q complement factor-engaging polypeptide as described herein. In some embodiments, the complement factor-engaging domain binds to C1q complement factor with a binding affinity of about 0.1 μM to about 2 μM, and the antigen-binding moiety binds to the target antigen with a binding affinity of less than 0.1 μM, as determined by biolayer interferometry. In some embodiments, the bispecific construct binds 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. In some embodiments, the complement factor-engaging domain has a binding affinity to the C1q complement factor of from about 150 nM to about 1 μM, as determined by biolayer interferometry. In some embodiments, the complement factor-engaging domain modulates the activity of a complement system by directly or indirectly activating the complement system. In some embodiments, the complement system is human complement system. In some embodiments, the complement factor-engaging domain directly binds a C1q complement factor. In some embodiments, the complement factor-engaging domain indirectly binds a C1q complement factor. In some embodiments, the C1q complement factor is human C1q complement factor. In some embodiments, the complement factor-engaging domain and the antigen-binding moiety are linked by a polypeptide linker sequence. In some embodiments, the polypeptide linker sequence comprises at least 5 amino acids or at least 30 amino acids. In some embodiments, the polypeptide linker sequence comprises about 10 amino acids to about 20 amino acids. In some embodiments, the polypeptide linker sequence comprises an amino acid sequence of any one of SEQ ID NO: 46– SEQ ID NO: 62. In some embodiments, the bispecific construct further comprises an albumin binding nanobody (Nb). In some embodiments, the albumin binding Nb is linked to the bispecific construct directly or indirectly at a N-terminal of the complement factor-engaging domain, between a C-terminal of the complement factor-engaging domain and a N-terminal of the antigen- binding moiety or a C-terminal of the antigen binding moiety. In some embodiments, the target antigen is a protein implicated in a disease. In some embodiments, the disease is a cancer, an autoimmune disease, an inflammatory disease, or a neurological disease. In some embodiments, the target antigen is a protein is expressed on the surface of a cell. In some embodiments, the cell is a cancer cell, an immune cell, an endothelial cell, an epithelial cell, or a microbial cell. In some embodiments, the cell is a cancer cell, and wherein the cancer cell is a carcinoma cell, a breast cancer cell, an ovarian cancer cell, a gastric cancer cell, or a colon cancer cell. In someembodiments, the cell is an immune cell, and wherein the immune cell is a natural killer (NK) cell, a macrophage, a monocyte, a neutrophil, a dendritic cell, a T cell, a B cell, a mast cell, a basophil, or an eosinophil. In some embodiments, the antigen-binding moiety binds to a cancer-specific marker, an immune-specific marker, a pathogen-specific marker, a tissue-specific marker, or an organ-specific marker. In some embodiments, the antigen-binding moiety binds to EGFR, CD38, CD19, CD20, CD55, CD59, CD7, HER2, EpCAM, or FOLR1. In some embodiments, the antigen- binding moiety binds to CD19. In some embodiments, the antigen-binding moiety binds to CD20. In some embodiments, the antigen-binding moiety binds to CD38. In some embodiments, the antigen-binding moiety binds to a cancer-specific marker, and wherein the cancer specific marker is EGFR.

[0019] In one embodiments the invention relates to an antibody or antibody construct comprising two antibody heavy chains (with each heavy chain comprising a VH domain, a CH1 domain and an FC portion, with the Fc portion being comprised of a CH2 domain and a CH3 domain) and two antibody light chains (with each light chain comprising a VL and a CL domain), which antibody or antibody construct comprises a complement factor-engaging domain antibody with reduced affinity for C1q (as further described herein). Said complement factor-engaging domain antibody with reduced affinity for C1q may be linked to one of the light chains or to one of the heavy chains, and may be linked to the N-terminus or the C-terminus of such heavy chain or light chain.

[0020] Without limiting the invention in any way, in certain aspects in which the invention may be put into practice (for example, and without limitation, as illustrated by the non-limiting examples of such heavy chains and light chains that are used in the Experimental Part), when a C1q binder is fused or linked to a heavy chain, it will be linked to the N-terminus of such heavy chain, and when a C1q binder is fused or linked to a light, it will be linked to the C-terminus of such light chain. Also, again without limiting the invention in any way, in certain aspects in which the invention may be put into practice (for example, and without limitation, as illustrated by the non-limiting examples of such heavy chains and light chains that are used in the Experimental Part), when a C1q binder is fused or linked to a the N-terminus of a heavy or light chain, it may for example be a suitable variant of Nb75 (as further described herein), and when a C1q binder is fused or linked to a the C-terminus of a heavy or light chain, it may for example be a suitable variant of Nb78. Also, again without limiting the invention in any way, in certain aspects in which the invention may be put into practice (for example, and without limitation, as illustrated by the non-limiting examples of such heavy chains and light chains that are used in the Experimental Part), when a C1q binder is fused or linked to a heavy chain, it may for example be a suitablevariant of Nb75 (as further described herein), and when a C1q binder is fused or linked to a light chain, it may for example be a suitable variant of Nb78.

[0021] In one embodiment, the invention relates to an antibody heavy chain (comprising a VH domain, a CH1 domain and an FCportion, with the Fc portion being comprised of a CH2 domain and a CH3 domain), which antibody heavy chain is fused linked (in particular at the N-terminus or C-terminus of the heavy chain) to a complement factor-engaging domain antibody with reduced affinity for C1q (as further described herein). Said antibody heavy chain may also comprise one or more mutations in the Fc portion (as further described herein). In a further embodiment, the invention relates to an antibody or antibody construct comprising such an antibody heavy chain.

[0022] In another embodiment, the invention relates to an antibody light chain (comprising a VLdomain and a CL domain), which antibody light chain is fused linked (in particular at the N- terminus or C-terminus of light chain) to a complement factor-engaging domain antibody with reduced affinity for C1q (as further described herein). In a further embodiment, the invention relates to an antibody or antibody construct comprising such an antibody light chain.

[0023] In some embodiments, the invention relates to an antibody or antibody construct comprising two antibody heavy chains (with each heavy chain comprising a VH domain, a CH1 domain and an FCportion, with the Fc portion being comprised of a CH2 domain and a CH3 domain) and two antibody light chains (with each light chain comprising a VL and a CL domain), which antibody or antibody construct comprises two complement factor-engaging domain antibodies with reduced affinity for C1q (as further described herein). Usually in practice, and preferably, the two complement factor-engaging domain antibodies with reduced affinity for C1q are fused to the two heavy chains (i.e. with one of the complement factor-engaging domain antibodies being fused to one of the heavy chains, and the other being fused to the other heavy chain), to the two light chains (i.e. with one of the complement factor-engaging domain antibodies being fused to one of the light chains, and the other being fused to the other light chain), or to one of the heavy chains and one of the light chains (i.e. with one of the complement factor-engaging domain antibodies being fused to one of the heavy chains, and the other being fused to one of the light chains).

[0024] According to one specific but non-limiting aspect, when a complement factor-engaging domain with reduced affinity for C1q is linked to a heavy chain, it is preferably linked to the N- terminus of such heavy chain.

[0025] According to another specific but non-limiting aspect, when a complement factor- engaging domain with reduced affinity for C1q is linked to a light chain, it is preferably linked to the C-terminus of such light chain (i.e., to the C-terminus of the of the CL domain of the light chain).

[0026] In another embodiment, the invention relates to an antibody comprising two antibody heavy chains and two antibody light chains, in which at least one of the heavy chains (and preferably both of the heavy chains) is linked or fused to a complement factor-engaging domain antibody with reduced affinity for C1q (as further described herein), in which the complement factor-engaging domain antibody is preferably linked or fused to the N-terminus of the antibody heavy chain (and when two C1q binders are present, both are linked to the N-terminus of the antibody heavy chain).

[0027] In another embodiment, the invention relates to an antibody comprising two antibody heavy chains and two antibody light chains, in which at least one of the light chains (and preferably both of the light chains) is linked or fused to a complement factor-engaging domain antibody with reduced affinity for C1q (as further described herein), in which the complement factor-engaging domain antibody is preferably linked or fused to the C-terminus (i.e. to the C-terminus of the CL domain) of the antibody light chain (and when two C1q binders are present, both are linked to the N-terminus of the antibody heavy chain).

[0028] In each of these antibodies of the invention, when the two complement factor-engaging domain antibodies, said two complement factor-engaging domain antibodies may be the same or different (and in practice, will usually be the same complement factor-engaging domain antibody).

[0029] Also, in these antibodies of the invention, the two heavy chains may be the same or different, but usually are the same; and the two light chains may be the same or different, but usually are the same. Usually, in the antibodies of the invention, the two heavy chains are the same and the two light chains are the same.

[0030] According to a particular but non-limiting aspect, the C1q binders are variants of Nb78 or Nb75 with reduced affinity for C1q (as further described herein), and in particular humanized variants of Nb78 or Nb75 with reduced affinity for C1q (as further described herein).

[0031] In a specific but non-limiting embodiment, the C1q binder may be chosen from the C1q binders in Table F, G or H that have an affinity for C1q within the ranges specified herein.

[0032] In some embodiments, the antigen-binding moiety comprises a Fc region, wherein the Fc region comprises one or more amino acid modification(s) with respect to a wildtype Fc region that improves the serum half-life as compared to the wildtype Fc region. In some embodiments, the one or more amino acid modification(s) is selected from the group consisting of M252Y, S254T, T256E, M428L, N434S, T256D, T307R, Q311V, N315D, N286D, T307R, H285N, and T307Q. In some embodiments, the modified antigen-binding Fc comprises a set of amino acid modifications selected from the group consisting of M252Y / S254T / T256E, M428L / N434S, T256D / T307R / Q311V, T256D / N315D / A378V, T256D / N286D / T307R / Q311V,H285N / T307Q / N315D, T256D / T307R / Q311V / A378V, H285D / Q311V / A378V, T256D / H285D / A378V, T256D / Q311V / A378V, T256D / H285D / N286D / T307R / A378V, T256D / H286D / T307R / Q311V / A378V, T307Q / Q311V / A378V, H285D / T307Q / A378V, and T256D / H285D / T307R / Q311V / A378V. Other suitable Fc mutations (and suitable combinations of Fc mutations will be clear to the skilled person based on the disclosure herein and in the co- pending International application and / or can easily be determined by the skilled person (optionally after a limited degree of trial and error). In some embodiments, the polypeptide construct further comprises an albumin-binding Nb. In some embodiments, the albumin-binding Nb is directly or indirectly linked to a N-terminus of the first complement factor-engaging domain or a N-terminus of the second complement factor-engaging domain. In some embodiments, the albumin-binding Nb is directly or indirectly linked to a C-terminus of a light chain or a C-terminus of a heavy chain in the antigen-binding moiety. In some embodiments, the albumin-binding Nb is directly or indirectly linked to a C-terminus of a light chain or a heavy chain in the Fab that binds CD38. In some embodiments, the polypeptide construct comprising the albumin-binding Nb has increased serum half-life as compared to the same polypeptide construct that lacks the albumin-binding Nb. In some embodiments, the polypeptide construct induces complement activation in the fluid phase as assessed by measuring generation of C4a. In some embodiments, the polypeptide construct does not induce complement activation in the fluid phase as assessed by measuring generation of C4a. In some embodiments, the polypeptide construct comprises an IgG region comprising a Fab region having the sequence of any one of SEQ ID NO: 40 – SEQ ID NO: 78. In some embodiments, the polypeptide construct comprises an IgG region comprising the sequence of any one of SEQ ID NO: 40 – SEQ ID NO: 78.

[0033] According to one specifically preferred but non-limiting embodiment, a polypeptide construct provided by the invention is in the form of an “antibody of the invention”, as further described herein. As will be clear to the skilled person, when a polypeptide construct provided by the invention is in the form of an antibody of the invention, such a construct will generally comprise four polypeptide chains (i.e. two antibody heavy chains and two antibody light chains) which chains suitably form the structure that is characteristic of a conventional four-chain antibody (but with two C1q binders fused or linked to said antibody structure, as further described herein). As also described herein, such 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 mutationsthat 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 I; and for illustration purposes only Table J gives examples of such non-naturally occurring Fc regions (using the Fc of antibody 11B8 mentioned herein 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. 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 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.

[0034] 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. Reference is again made to Tables I and J. Again for illustration purposes only, Table J also gives a heavy chain sequence with a non-naturally occurring Fc domain (SEQ ID NO: 256, which is based on the heavy chain sequence of 11B8), two of which can for example be suitably combined with two of the light chains of SEQ ID NO: 257 (with each such light chain comprising a C1q binder as described herein) in order to provide an antibody of the invention).

[0035] 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 F; and for illustration purposes only Table G gives examples of such non- naturally occurring Fc regions (using the Fc of antibody 11B8 mentioned herein as a reference sequence, again for illustration purposes only). Such mutations may also be used in a suitablecombination, as will be clear to the skilled person. 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 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.

[0036] 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. Reference is again made to Tables F and G. Again for illustration purposes only, Table G also gives a heavy chain sequence with a non-naturally occurring Fc domain (SEQ ID NO: 269, which is based on the heavy chain sequence of 11B8), two of which can for example be suitably combined with two of the light chains of SEQ ID NO: 270 (with each such light chain comprising a C1q binder as described herein) in order to provide an antibody of the invention).

[0037] In this respect, it should be noted that, while antibodies of the invention as described herein will generally have one or more of the following advantages: − improved ability to “activate complement on” a cell that expresses the antigen 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 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 the relevant antigen (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 the relevant antigen (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.

[0038] Also disclosed herein are pharmaceutical compositions that comprise a C1q complement factor-engaging polypeptide as described herein or a bispecific construct as described herein.

[0039] Also disclosed herein is a composition that comprises a bispecific construct that comprises: (a) a complement factor-engaging domain that binds to a C1q complement factor with a binding affinity of about 0.1 μM, as determined by biolayer interferometry; and (b) an antigen-binding moiety linked to the complement factor-engaging domain, wherein the antigen-binding moiety binds to an antigen expressed by a cell with a binding affinity of less than 0.1 μM, as determined by biolayer interferometry. In some embodiments, the bispecific construct is a bispecific construct as described herein. In some embodiments, the complement factor-engaging domain is a complement factor-engaging domain as described herein, wherein the complement factor- engaging domain binds to a C1q complement factor with a binding affinity of about 0.1 μM, as determined by biolayer interferometry. In some embodiments, the complement factor-engaging domain binds to a C1q complement factor with a binding affinity of 0.08 μM to 0.12 μM, or 0.09 μM to 0.11 μM, as determined by biolayer interferometry. In some embodiments, the antigen- binding moiety is an antigen-binding moiety as described herein, wherein the antigen-binding moiety binds to an antigen expressed by a cell with a binding affinity of less than 0.1 μM, as determined by biolayer interferometry. In some embodiments, the antigen is a cancer-specific marker, an immune-specific marker, an organ-specific marker, or a protein implicated in a disease. In some embodiments, the antigen is a cancer specific marker selected from the group consisting of: CD7, CD19, CD20, CD38, EGFR, EpCAM, FOLR1 and HER2. In some embodiments, thebispecific construct further comprises an albumin-binding moiety. In some embodiments, the albumin-binding moiety is an albumin-binding moiety as described herein.

[0040] According to one specifically preferred but non-limiting embodiment, a polypeptide construct provided by the invention is in the form of an “antibody of the invention”, as further described herein. As will be clear to the skilled person, when a polypeptide construct provided by the invention is in the form of an antibody of the invention, such a construct will generally comprise four polypeptide chains (i.e. two antibody heavy chains and two antibody light chains) which chains suitably form the structure that is characteristic of a conventional four-chain antibody (but with at least one and preferably two C1q binders with reduced affinity for C1q (as described herein) fused or linked to said antibody structure, as further described herein). As also described herein, such 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).

[0041] According to one specifically preferred but non-limiting embodiment, a polypeptide construct provided by the invention comprises a single polypeptide chain that comprises at least one C1q binder with reduced affinity (as described herein) and the antigen binding domain (and optionally other suitable binding domains or binding units), which are suitably fused or linked to each other (in each case, and independently of each other, directly or via a suitable linker). It will be clear to the skilled person that, in this embodiment, the C1q binder and the antigen binding domain (and often also the other binding domains or binding units) will all be single domain antibodies (as described herein). In particular, but without limitation, the C1q binder and the antigen binding domain (and often also the other binding domains or binding units) may all be Nanobodies (as further described herein, and including VHH domains or ISVDs that have been derived from VHH domains). In this embodiment, the polypeptide construct provided by the invention may also comprise a binding domain or binding unit that provides the polypeptide construct with a desired and / or increased half-life. Such binding domain or binding unit will be clear to the skilled person and may in particular, but without limitation, include a binding domain or binding unit that can bind to serum albumin (for example and without limitation, a Nanobody that can bind to serum albumin, for which reference is for example made to WO 08 / 020079. According to a specific but non-limiting aspect, when a polypeptide construct provided by the invention comprises a single polypeptide chain, it may in particular be a (in the form of) a “bispecific complement engager” or “BiCE” (for which reference is made to Pedersen et al., supra), and in particular (in the form of) a BiCE that comprises a C1q binder with reduced affinityfor C1q (as further described herein) in which said C1q binders are preferably as further described in International application entitled “Engineered complement engaging polypeptides”.

[0042] Also disclosed herein is a method of enhancing anti-cancer effect of an antibody, the method comprising fusing the antibody to a complement factor-engaging domain, wherein the complement factor-engaging domain has a lower affinity for a complement factor as compared to an affinity of the antibody to an antigen. In some embodiments, the antibody or fragment thereof has a sequence of any one of SEQ ID NO: 63 – SEQ ID NO: 99. In some embodiments, the complement factor-engaging domain is a complement factor-engaging domain as described herein. In some embodiments, the antigen is the target antigen of the antibody. In some embodiments, the target antigen is a target protein as described herein. In some embodiments, the target antigen is a cancer marker as described herein.

[0043] Also disclosed herein is a method of enhancing anti-inflammatory effect of an antibody or fragment thereof, the method comprising fusing the antibody or fragment thereof to a complement factor-engaging domain, wherein the complement factor-engaging domain has a lower affinity for a complement factor as compared to an affinity of the antibody or fragment thereof to an antigen. In some embodiments, the antibody or fragment thereof has a sequence of any one of SEQ ID NO: 63 – SEQ ID NO: 99. In some embodiments, the complement factor-engaging domain is a complement factor-engaging domain as described herein. In some embodiments, the antigen is the target antigen of the antibody. In some embodiments, the target antigen is a target protein as described herein. In some embodiments, the antigen is an inflammatory marker. In some embodiments, the target antigen is an autoimmune marker or a cancer marker as described herein.

[0044] Also disclosed herein is a composition for use in inducing complement-mediated cancer cell death when contacted with a cancer cell, the composition comprising a bispecific construct that comprises: (i) a complement factor-engaging domain that binds to a human C1q complement factor with a binding affinity of about 1 μM, and (ii) an antigen binding moiety that binds to a cancer-specific marker expressed by a cancer cell; wherein contacting of the composition with the cancer cell that expresses the cancer-specific marker results in complement-mediated cell death of the cancer cell that expresses the cancer-specific marker. In some embodiments, the bispecific construct is a bispecific construct as described herein. In some embodiments, the complement factor-engaging domain is a complement factor-engaging domain as described herein, wherein the complement factor-engaging domain binds to a human C1q complement factor with a binding affinity of about 1 μM. In some embodiments, the complement factor-engaging domain binds to human C1q complement factor with a binding affinity of about 1 μM, as determined by biolayer interferometry. In some embodiments, the complement factor-engaging domain binds to a C1qcomplement factor, such as a human C1q complement factor, with a binding affinity of 0.9 μM to 1.1 μM, or 0.95 μM to 1.05 μM, or 0.99 μM to 1.01 μM, as determined by biolayer interferometry. In some embodiments, the antigen binding moiety is an antigen binding moiety as described herein, wherein the antigen binding moiety binds to a cancer-specific marker expressed by a cancer cell. In some embodiments, the cancer specific marker is a cancer marker as described herein.

[0045] Also disclosed herein is a method of inducing complement-mediated cancer cell death, the method comprising: contacting a cancer cell with a bispecific construct that comprises: (i) a complement factor-engaging domain that binds to a human C1q complement factor with a binding affinity of about 1 μM, and (ii) an antigen binding moiety that binds to a cancer-specific marker expressed by a cancer cell; wherein the contacting results in complement-mediated cell death of the cancer cell that expresses the cancer-specific marker. In some embodiments, the bispecific construct is a bispecific construct as described herein. In some embodiments, the complement factor-engaging domain is a complement factor-engaging domain as described herein, wherein the complement factor-engaging domain binds to a human C1q complement factor with a binding affinity of about 1 μM In some embodiments, the complement factor-engaging domain binds to human C1q complement factor with a binding affinity of about 1 μM, as determined by biolayer interferometry In some embodiments, the complement factor-engaging domain binds to a C1q complement factor, such as a human C1q complement factor, with a binding affinity of 0.9 μM to 1.1 μM, or 0.95 μM to 1.05 μM, or 0.99 μM to 1.01 μM, as determined by biolayer interferometry. In some embodiments, the antigen binding moiety is an antigen binding moiety as described herein, wherein the antigen binding moiety binds to a cancer-specific marker expressed by a cancer cell. In some embodiments, the cancer specific marker is a cancer marker as described herein.

[0046] Also disclosed herein is a composition for use in treating an autoimmune disease in a subject, wherein the composition comprises a bispecific construct that comprises: (a) a complement factor-engaging domain that binds to human C1q complement factor with a binding affinity of about 1 μM, as determined by biolayer interferometry (BLI); and (b) an antigen-binding moiety that binds to an autoimmune marker expressed by a cell. In some embodiments, the bispecific construct is a bispecific construct as described herein. In some embodiments, the complement factor-engaging domain is a complement factor-engaging domain as described herein, wherein the complement factor-engaging domain binds to a human C1q complement factor with a binding affinity of about 1 μM, as determined by biolayer interferometry In some embodiments, the complement factor-engaging domain binds to a C1q complement factor, such as a human C1q complement factor, with a binding affinity of 0.9 μM to 1.1 μM, or 0.95 μM to 1.05 μM, or 0.99 μM to 1.01 μM, as determined by biolayer interferometry. In some embodiments, the antigenbinding moiety is an antigen binding moiety as described herein, wherein the antigen binding moiety binds to an autoimmune marker expressed by a cell. In some embodiments, the autoimmune marker is an autoimmune marker as described herein. In some embodiments, the autoimmune disease is multiple sclerosis, psoriasis, psoriatic arthritis, rheumatoid arthritis, systemic lupus erythematosus, Crohn's disease, Sjogren's syndrome, Behcet's disease, ulcerative colitis, or Guillain-Barre syndrome.

[0047] Also disclosed herein is a method of treating an autoimmune disease in a subject in need thereof, the method comprising administering to the subject a bispecific construct that comprises: (a) a complement factor-engaging domain that binds to human C1q complement factor with a binding affinity of about 1 μM, as determined by biolayer interferometry (BLI); and (b) an antigen- binding moiety that binds to an autoimmune marker expressed by a cell; wherein the administering is sufficient to treat the autoimmune disease in the subject. In some embodiments, the bispecific construct is a bispecific construct as described herein. In some embodiments, the complement factor-engaging domain is a complement factor-engaging domain as described herein, wherein the complement factor-engaging domain binds to a human C1q complement factor with a binding affinity of about 1 μM, as determined by biolayer interferometry In some embodiments, the complement factor-engaging domain binds to a C1q complement factor, such as a human C1q complement factor, with a binding affinity of 0.9 μM to 1.1 μM, or 0.95 μM to 1.05 μM, or 0.99 μM to 1.01 μM, as determined by biolayer interferometry. In some embodiments, the antigen binding moiety is an antigen binding moiety as described herein, wherein the antigen binding moiety binds to an autoimmune marker expressed by a cell. In some embodiments, the autoimmune marker is an autoimmune marker as described herein. In some embodiments, the autoimmune disease is multiple sclerosis, psoriasis, psoriatic arthritis, rheumatoid arthritis, systemic lupus erythematosus, Crohn's disease, Sjogren's syndrome, Behcet's disease, ulcerative colitis, or Guillain-Barre syndrome.

[0048] Also disclosed herein is a composition for use in treating cancer characterized by aberrant expression of a cancer-specific marker by a cancer cell in a subject in need thereof, wherein the composition comprises: (a) a complement factor-engaging domain that binds to human C1q complement factor with a binding affinity of about 1 μM, as determined by biolayer interferometry; and (b) an antigen-binding moiety that binds to the cancer specific marker expressed by the cancer cell. In some embodiments, the complement factor-engaging domain and the antigen-binding domain are provided as a bispecific construct as described herein. In some embodiments, the complement factor-engaging domain is a complement factor-engaging domain as described herein, wherein the complement factor-engaging domain binds to a human C1qcomplement factor with a binding affinity of about 1 μM, as determined by biolayer interferometry In some embodiments, the complement factor-engaging domain binds to a C1q complement factor, such as a human C1q complement factor, with a binding affinity of 0.9 μM to 1.1 μM, or 0.95 μM to 1.05 μM, or 0.99 μM to 1.01 μM, as determined by biolayer interferometry. In some embodiments, the antigen binding moiety is an antigen binding moiety as described herein, wherein the antigen binding moiety binds to a cancer-specific marker expressed by a cancer cell. In some embodiments, the cancer specific marker is a cancer marker as described herein. In some embodiments, the cancer-specific marker is selected from the group consisting of CD7, CD19, CD20, CD38, CD55, CD59, EGFR, EpCAM, FOLR1, and HER2.

[0049] Also disclosed herein is a method of treating cancer characterized by aberrant expression of a cancer-specific marker by a cancer cell in a subject in need thereof, the method comprising administering to the subject a bispecific construct that comprises: (a) a complement factor- engaging domain that binds to human C1q complement factor with a binding affinity of about 1 μM, as determined by biolayer interferometry; and (b) an antigen-binding moiety that binds to the cancer specific marker expressed by the cancer cell; wherein the administering is sufficient to treat the cancer in the subject. In some embodiments, the bispecific construct is a bispecific construct as described herein. In some embodiments, the complement factor-engaging domain is a complement factor-engaging domain as described herein, wherein the complement factor- engaging domain binds to a human C1q complement factor with a binding affinity of about 1 μM, as determined by biolayer interferometry In some embodiments, the complement factor-engaging domain binds to a C1q complement factor, such as a human C1q complement factor, with a binding affinity of 0.9 μM to 1.1 μM, or 0.95 μM to 1.05 μM, or 0.99 μM to 1.01 μM, as determined by biolayer interferometry. In some embodiments, the antigen binding moiety is an antigen binding moiety as described herein, wherein the antigen binding moiety binds to a cancer-specific marker expressed by a cancer cell. In some embodiments, the cancer specific marker is a cancer marker as described herein. In some embodiments, the cancer-specific marker is selected from the group consisting of CD7, CD19, CD20, CD38, CD55, CD59, EGFR, EpCAM, FOLR1, and HER2.

[0050] Also disclosed herein is a composition for use in treating cancer characterized by aberrant expression of CD19 by a cancer cell, where the composition comprises a bispecific construct that comprises: (a) a complement factor-engaging domain that binds to human C1q complement factor with a binding affinity of about 1 μM, as determined by biolayer interferometry; and (b) an antigen-binding moiety that binds to CD19 expressed on the cancer cell. In some embodiments, the bispecific construct is a bispecific construct as described herein. In some embodiments, the complement factor-engaging domain is a complement factor-engaging domain as described herein,wherein the complement factor-engaging domain binds to a human C1q complement factor with a binding affinity of about 1 μM, as determined by biolayer interferometry In some embodiments, the complement factor-engaging domain binds to a C1q complement factor, such as a human C1q complement factor, with a binding affinity of 0.9 μM to 1.1 μM, or 0.95 μM to 1.05 μM, or 0.99 μM to 1.01 μM, as determined by biolayer interferometry. In some embodiments, the antigen binding moiety is an antigen binding moiety as described herein, wherein the target antigen is CD19.

[0051] Also disclosed herein is a method of treating cancer characterized by aberrant expression of CD19 by a cancer cell in a subject in need thereof, wherein the method comprises administering to the subject a bispecific construct that comprises: (a) a complement factor-engaging domain that binds to human C1q complement factor with a binding affinity of about 1 μM, as determined by biolayer interferometry; and (b) an antigen-binding moiety that binds to CD19 expressed on the cancer cell; wherein the administering is sufficient to treat the cancer in the subject. In some embodiments, the bispecific construct is a bispecific construct as described herein. In some embodiments, the complement factor-engaging domain is a complement factor-engaging domain as described herein, wherein the complement factor-engaging domain binds to a human C1q complement factor with a binding affinity of about 1 μM, as determined by biolayer interferometry. In some embodiments, the complement factor-engaging domain binds to a C1q complement factor, such as a human C1q complement factor, with a binding affinity of 0.9 μM to 1.1 μM, or 0.95 μM to 1.05 μM, or 0.99 μM to 1.01 μM, as determined by biolayer interferometry. In some embodiments, the antigen binding moiety is an antigen binding moiety as described herein, wherein the target antigen is CD19.

[0052] Also disclosed herein is a composition for use in treating cancer characterized by aberrant expression of CD20 by a cancer cell in a subject in need thereof, the composition comprising a bispecific construct that comprises: (a) a complement factor-engaging domain that binds to human C1q complement factor with a binding affinity of about 1 μM, as determined by biolayer interferometry; and (b) an antigen-binding moiety that binds to CD20 expressed on the cancer cell. In some embodiments, the bispecific construct is a bispecific construct as described herein. In some embodiments, the complement factor-engaging domain is a complement factor-engaging domain as described herein, wherein the complement factor-engaging domain binds to a human C1q complement factor with a binding affinity of about 1 μM, as determined by biolayer interferometry In some embodiments, the complement factor-engaging domain binds to a C1q complement factor, such as a human C1q complement factor, with a binding affinity of 0.9 μM to 1.1 μM, or 0.95 μM to 1.05 μM, or 0.99 μM to 1.01 μM, as determined by biolayer interferometry.In some embodiments, the antigen binding moiety is an antigen binding moiety as described herein, wherein the target antigen is CD20.

[0053] Also disclosed herein is a method of treating cancer characterized by aberrant expression of CD20 by a cancer cell in a subject in need thereof, the method comprising administering to the subject a bispecific construct that comprises: (a) a complement factor-engaging domain that binds to human C1q complement factor with a binding affinity of about 1 μM, as determined by biolayer interferometry; and (b) an antigen-binding moiety that binds to CD20 expressed on the cancer cell; wherein the administering is sufficient to treat the cancer in the subject. In some embodiments, the bispecific construct is a bispecific construct as described herein. In some embodiments, the complement factor-engaging domain is a complement factor-engaging domain as described herein, wherein the complement factor-engaging domain binds to a human C1q complement factor with a binding affinity of about 1 μM, as determined by biolayer interferometry In some embodiments, the complement factor-engaging domain binds to a C1q complement factor, such as a human C1q complement factor, with a binding affinity of 0.9 μM to 1.1 μM, or 0.95 μM to 1.05 μM, or 0.99 μM to 1.01 μM, as determined by biolayer interferometry. In some embodiments, the antigen binding moiety is an antigen binding moiety as described herein, wherein the target antigen is CD20.

[0054] Also disclosed herein is a composition for use in treating cancer characterized by aberrant expression of CD38 by a cancer cell in a subject in need thereof, the composition comprising a bispecific construct that comprises: (a) a complement factor-engaging domain that binds to human C1q complement factor with a binding affinity of about 1 μM, as determined by biolayer interferometry; and (b) an antigen-binding moiety that binds to CD38 expressed on the cancer cell. In some embodiments, the bispecific construct is a bispecific construct as described herein. In some embodiments, the complement factor-engaging domain is a complement factor-engaging domain as described herein, wherein the complement factor-engaging domain binds to a human C1q complement factor with a binding affinity of about 1 μM, as determined by biolayer interferometry In some embodiments, the complement factor-engaging domain binds to a C1q complement factor, such as a human C1q complement factor, with a binding affinity of 0.9 μM to 1.1 μM, or 0.95 μM to 1.05 μM, or 0.99 μM to 1.01 μM, as determined by biolayer interferometry. In some embodiments, the antigen binding moiety is an antigen binding moiety as described herein, wherein the target antigen is CD38.

[0055] Also disclosed herein is a method of treating cancer characterized by aberrant expression of CD38 by a cancer cell in a subject in need thereof, the method comprising administering to the subject a bispecific construct that comprises: (a) a complement factor-engaging domain that bindsto human C1q complement factor with a binding affinity of about 1 μM, as determined by biolayer interferometry; and (b) an antigen-binding moiety that binds to CD38 expressed on the cancer cell; wherein the administering is sufficient to treat the cancer in the subject. In some embodiments, the bispecific construct is a bispecific construct as described herein. In some embodiments, the complement factor-engaging domain is a complement factor-engaging domain as described herein, wherein the complement factor-engaging domain binds to a human C1q complement factor with a binding affinity of about 1 μM, as determined by biolayer interferometry In some embodiments, the complement factor-engaging domain binds to a C1q complement factor, such as a human C1q complement factor, with a binding affinity of 0.9 μM to 1.1 μM, or 0.95 μM to 1.05 μM, or 0.99 μM to 1.01 μM, as determined by biolayer interferometry. In some embodiments, the antigen binding moiety is an antigen binding moiety as described herein, wherein the target antigen is CD38.

[0056] Also disclosed herein is a composition for use in treating cancer characterized by aberrant expression of EGFR by a cancer cell in a subject in need thereof, the composition comprising a bispecific construct that comprises: (a) a complement factor-engaging domain that binds to human C1q complement factor with a binding affinity of about 1 μM, as determined by biolayer interferometry; and (b) an antigen-binding moiety that binds to EGFR expressed on the cancer cell. In some embodiments, the bispecific construct is a bispecific construct as described herein. In some embodiments, the complement factor-engaging domain is a complement factor-engaging domain as described herein, wherein the complement factor-engaging domain binds to a human C1q complement factor with a binding affinity of about 1 μM, as determined by biolayer interferometry In some embodiments, the complement factor-engaging domain binds to a C1q complement factor, such as a human C1q complement factor, with a binding affinity of 0.9 μM to 1.1 μM, or 0.95 μM to 1.05 μM, or 0.99 μM to 1.01 μM, as determined by biolayer interferometry. In some embodiments, the antigen binding moiety is an antigen binding moiety as described herein, wherein the target antigen is EGFR.

[0057] Also disclosed herein is a method of treating cancer characterized by aberrant expression of EGFR by a cancer cell in a subject in need thereof, the method comprising administering to the subject a bispecific construct that comprises: (a) a complement factor-engaging domain that binds to human C1q complement factor with a binding affinity of about 1 μM, as determined by biolayer interferometry; and (b) an antigen-binding moiety that binds to EGFR expressed on the cancer cell; wherein the administering is sufficient to treat the cancer in the subject. In some embodiments, the bispecific construct is a bispecific construct as described herein. In some embodiments, the complement factor-engaging domain is a complement factor-engaging domain as described herein,wherein the complement factor-engaging domain binds to a human C1q complement factor with a binding affinity of about 1 μM, as determined by biolayer interferometry In some embodiments, the complement factor-engaging domain binds to a C1q complement factor, such as a human C1q complement factor, with a binding affinity of 0.9 μM to 1.1 μM, or 0.95 μM to 1.05 μM, or 0.99 μM to 1.01 μM, as determined by biolayer interferometry. In some embodiments, the antigen binding moiety is an antigen binding moiety as described herein, wherein the target antigen is EGFR. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Novel features of exemplary embodiments are set forth with particularity in the appended claims. A better understanding of the features and advantages will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosed systems and methods are utilized, and the accompanying drawings of which:

[0059] FIG. 1A – FIG. 1J depict binding affinities of exemplary bispecific single domain antibody constructs of the present disclosure to C1q.

[0060] FIG. 2A – FIG. 2B depict cytotoxicity of exemplary bispecific single domain antibody constructs of the present disclosure that bind CD38 and C1q. FIG. 2A depicts a schematic of the bispecific constructs as disclosed herein. FIG. 2B illustrates the cytotoxicity of exemplary constructs in CD38-expressing WSU-DLCL2 cells.

[0061] FIG. 3A – FIG. 3F depict binding affinities of exemplary humanized bispecific affinity variants of single domain antibody constructs of the present disclosure.

[0062] FIG. 4A – FIG. 4G show biolayer interferometry graphs for of binding affinities for exemplary humanized bispecific single domain antibody constructs of the present disclosure.

[0063] FIG.5A depicts cytotoxicity of exemplary bispecific single domain antibody constructs of the present disclosure that bind CD38 and C1q. The y-axis depicts percent cytotoxicity, and the x- axis depicts concentration (nM). FIG.5B shows a graph of the on-off rates of MU1053 binding to CD38.

[0064] FIG. 6 depict cytotoxicity of exemplary low and high affinity bispecific single domain antibody constructs of the present disclosure that bind CD38 and C1q compared to Daratumumab and Hexabody-38 (GEN3014, see Hiemstra et al., EBioMedicine.2023 Jul;93:104663). The y-axis depicts percent cytotoxicity, and the x-axis depicts concentration (nM).

[0065] FIG. 7 depicts the binding affinities of exemplary humanized bispecific single domain antibody constructs of the present disclosure.

[0066] FIG. 8 depict cytotoxicity of exemplary bispecific single domain antibody constructs of the present disclosure that bind CD38 and C1q in WSU-DLCL2 cells. The y-axis depicts percent cytotoxicity, and the x-axis depicts concentration (nM).

[0067] FIG.9A – FIG.9B compare binding affinity and cytotoxicity of exemplary low and high affinity bispecific single domain antibody constructs of the present disclosure that bind EGFR and C1q. FIG. 9A depicts the on and off rates of the bispecific single domain antibody. FIG. 9B illustrates the cytotoxicity of exemplary construct recited in MDA-MB-468 cells. The y-axis depicts percent cytotoxicity, and the x-axis depicts concentration (nM).

[0068] FIG. 10A- FIG. 10B compare binding affinity and cytotoxicity of exemplary bispecific single domain antibody constructs of the present disclosure that bind EGFR and C1q (FIG.10A) and CD38 and C1q (FIG. 10B). FIG. 10A illustrates the cytotoxicity of exemplary construct in MDA-MB-468 cells FIG.10B illustrates the cytotoxicity of exemplary construct in WSU-DLCL2 cells. The y-axis depicts percent cytotoxicity, and the x-axis depicts concentration (nM).

[0069] FIG.11A- FIG.11G show the binding affinities and cytotoxicity of exemplary bispecific single domain antibody constructs with different protein targets. FIG. 11A illustrates the cytotoxicity of an exemplary bispecific construct that binds C1q and CD38 in DND-41 cells. FIG. 11B illustrates the cytotoxicity of an exemplary bispecific construct that binds C1q and CD20 in Raji cells. FIG. 11C illustrates the cytotoxicity of an exemplary bispecific construct that binds C1q and CD19 in Raji cells. FIG. 11D illustrates the cytotoxicity of an exemplary bispecific construct that binds C1q and CD7 in DND-41 cells. FIG. 11E illustrates the cytotoxicity of an exemplary bispecific construct that binds C1q and FOLR1 in IGROV1 cells. FIG.11F illustrates the cytotoxicity of an exemplary bispecific construct that binds C1q and EpCAM in SW403 cells. FIG. 11G illustrates the cytotoxicity of an exemplary bispecific construct that binds C1q and HER2 in OE19 cells. FIG. 11H illustrates the cytotoxicity of an exemplary bispecific construct that binds C1q and EGFR in A431 cells.

[0070] FIG.12 shows a graph of the cytotoxicity of Daudi cells treated with CD19 mAb or CD19- BiCE at varying concentrations. The y-axis depicts percent cytotoxicity, and the x-axis depicts concentration (nM).

[0071] FIG.13 shows a graph of the cytotoxicity of Daudi cells treated with CD20 mAb, CD20- BiCE, or Rituximab at varying concentrations. The y-axis depicts percent cytotoxicity, and the x- axis depicts concentration (nM).

[0072] FIG. 14 shows a schematic of an exemplary CD20-BiCE and structural features of the polypeptide construct.

[0073] FIG.15 shows a graph the cytotoxicity of human B cells treated with CD20 mAb, CD20- BiCE, or CD-20 BiCE- LALAPG at varying concentrations. The y-axis depicts percent cytotoxicity, and the x-axis depicts concentration (nM).

[0074] FIG. 16 shows a graph of B cell depletion in human peripheral blood mononuclear cells (PBMCs) treated with CD20 mAb, CD20-BiCE, or CD-20 BiCE- LALAPG at varying concentrations. The y-axis depicts percent B cell killing, and the x-axis depicts concentration (nM).

[0075] FIG. 17 shows a graph of antibody-dependent cellular cytotoxicity (ADCC) in human peripheral plood mononuclear cells (PBMCs) treated with CD20 mAb, CD20-BiCE, or CD-20 BiCE- LALAPG at varying concentrations. The y-axis depicts percent cell lysis, and the x-axis depicts concentration (nM).

[0076] FIG. 18 shows a graph of a human whole blood assay for B cell killing in human blood treated with CD20 mAb, CD20-BiCE, CD-20 BiCE- LALAPG, alemtuzumab, and cetuximab at varying concentrations. The y-axis depicts percent B cell killing, and the x-axis depicts concentration (nM).

[0077] FIG. 19 shows a schematic of Non-Human Primate (NHP) evaluation, conditions, blood draw schedule, and biopsy schedule.

[0078] FIG. 20 shows a graph of CD19+ and CD20+ blood cells determined by flow cytometry in NHPs treated with CD20-BiCE (sample #: 1001, 1002, 1003) or CD20-BiCE-LALAPG (sample #: 2001, 2002, 2003). The y-axis depicts the percentage of CD19 positive or CD20 positive cells as indicated, and the x-axis depicts time in days.

[0079] FIG.21 shows a graph of CD19+ cells determined by flow cytometry in NHP lymphnodes following treatment with CD20-BiCE (sample #: 1001, 1002, 1003) or CD20-BiCE-LALAPG (sample #: 2001, 2002, 2003). The y-axis depicts the percentage of CD19 positive cells, and the x-axis depicts time in days.

[0080] FIG. 22 shows a graph of the cytotoxicity of NHP B cell populations treated with CD20 mAb, CD20-BiCE, or CD-20 BiCE- LALAPG at varying concentrations in vitro. The y-axis depicts percent cytotoxicity, and the x-axis depicts concentration (nM).

[0081] FIG. 23A- FIG. 23B show graphs of B cell killing in populations of PBMCs using different concentrations of CD-20 mAb, CD20-BiCE, and CD20-BiCE-LALAPG. FIG. 23A shows a graph of B cell killing in a population of non-naïve PBMCs with matched serum. FIG. 23B shows a graph of B cell killing in a population of naiive PBMCs with matched serum. The percentage of B cells killed following administration to the cells was quantified for each concentration. The y-axis depicts percent B cell killing, and the x-axis depicts concentration (nM).

[0082] FIG. 24 shows a schematic of Dara-BiCE, HexaBody-CD38, daratumumab, and a BiCE negative control antibody.

[0083] FIG. 25 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.

[0084] FIG.26 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).

[0085] FIG.27 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.

[0086] FIG. 28 shows a heat map of protein levels and max cell lysis of cell lines treated with Dara-IgG BiCE, Daratumumab, or the CD38 hexabody.

[0087] FIG.29 shows a graph of cytotoxicity of DOHH2 cells treated with CM91-751; CM1253- 1255 (Felzartamab); CM1267-1269 (mezagitamab); CM 1781-1782 (CM313); CM1781-1783 (CM313 + C1q binder); daratumumamb; CM220-221 (CD28 Hexabody); or isatuximab. The y- axis depicts percent cytotoxicity, and the x-axis depicts concentration (nM).

[0088] FIG. 30 shows a graph of cytotoxicity of WSU cells treated with CM1232-1234 (daratumumamb 005); CM 1233-1234; 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).

[0089] FIG. 31 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).

[0090] FIG. 32 shows a graphs of the cytotoxic effects of C1q and CD38-targeting polypeptide constructs (BiCE constructs) relative to their parental monoclonal antibody. The y-axis depicts percent cytotoxicity, and the x-axis depicts concentration (nM).

[0091] FIG. 33 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).

[0092] FIG.34 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.

[0093] FIG. 35A-FIG. 35D 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.

[0094] FIG.36 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.

[0095] FIG. 37 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 Definitions

[0096] Unless otherwise indicated, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Reference is for example made to the standard handbooks mentioned in paragraph a) on page 46 of WO 08 / 020079 of Ablynx N.V. entitled “Amino acid sequences directed against IL-6R and polypeptides comprising the same for the treatment of diseases and disorders associated with Il-6 mediated signalling”, as well as background publications and handbooks cited therein. Unless otherwise indicated or obvious from context, the following terms have the following meanings:

[0097] Unless indicated otherwise, all methods, steps, techniques and manipulations that are not specifically described in detail can be performed and have been performed in a manner known per se, as will be clear to the skilled person. Reference is for example again made to the standard handbooks and the general background art mentioned herein and to the further references cited therein; as well as to for example the following reviews Presta, Adv. Drug Deliv. Rev. 2006, 58 (5-6): 640-56; Levin and Weiss, Mol. Biosyst. 2006, 2(1): 49-57; Irving et al., J. Immunol. Methods, 2001, 248(1-2), 31-45; Schmitz et al., Placenta, 2000, 21 Suppl. A, S106-12, Gonzales et al., Tumour Biol., 2005, 26(1), 31-43, which describe techniques for protein engineering, such as affinity maturation and other techniques for improving the specificity and other desired properties of proteins such as immunoglobulins.

[0098] Amino acid residues will be indicated according to the standard three-letter or one-letter amino acid code. Reference is made to Table A-2 on page 48 of the International Application No. WO 2008 / 020079.

[0099] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a sample” includes a plurality of samples, including mixtures thereof.

[0100] Any reference to “or” herein is intended to encompass “and / or” unless otherwise stated. As used herein, the term “and / or” includes any and all combinations of one or more of the associated listed items.

[0101] Use of the term “including” as well as other forms, such as “includes” and “included,” is not limiting.

[0102] As used herein, the term “comprise” and its grammatical equivalents specifies the presence of stated features, integers, steps, operations, elements, and / or components, but does not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0103] As used herein, the term “about” in reference to a number or range of numbers is understood to mean the stated number and numbers + / - 10% thereof, or 10% below the lower listed limit and 10% above the higher listed limit for the values listed for a range, as well as variations of ±5%, ±1%, ±0.5%, or even ±0.1%.

[0104] Unless indicated otherwise, the terms “immunoglobulin sequence”, “sequence”, “nucleotide sequence” and “nucleic acid” are as described in paragraph b) on page 46 of WO 08 / 020079.

[0105] The term “protein”, “peptide” and “polypeptide” are used interchangeably to refer to an oligomer of two or more linked amino acids or derivatives of the same.

[0106] The term “antibody” refers to a polypeptide or protein capable of recognizing and binding an antigen comprising at least one antigen binding site. Said antigen binding site preferably comprises at least one complementarity determining region (CDR). The present disclosure relates primarily to single domain antibodies.

[0107] The terms “single domain antibodies”, “single variable domains” and “immunoglobulin single variable domain” (or “ISVD”), which terms are used interchangeably herein, have their understood meaning in the art and generally refer to a binding domain that comprises an immunoglobulin fold and that can form a functional antigen binding site without having to pair with another immunoglobulin-based binding domain (as is for example the case with the VH and VL domains that are present in conventional 4-chain antibodies, which need to pair with another VL or VH domain, respectively, to form a functional antigen binding site). Accordingly, single domain antibodies, single variable domains, and immunoglobulin single variable domains / ISVDs will generally comprise only one single domain or fragment of a domain of a whole antibody. Thesingle domain may be a heavy chain constant region (CH), a heavy chain variable region (VH), a light chain constant region (CL) or a light chain variable region (VL) or a fragment thereof. In a preferred embodiment the single domain is a heavy chain variable region (VH). Single domain antibodies typically have molecular weights in the range of 12–15 kDa, i.e., much lower than common antibodies, ranging typically from 150 to 160 kDa. Single domain antibodies are also smaller than Fab fragments (~50 kDa) of heterotetrameric antibodies comprising one light chain and half a heavy chain. In some embodiments, the single domain antibody is derived from a heavy chain antibody such as a camelid antibody or a cartilaginous fish heavy chain antibody. In some embodiments, the single domain antibody comprises a variable domain of heavy chain antibody (VHH domain), such as a VHH domain as described herein. In some embodiments, the single domain antibody comprises a VNAR domain, such as a VNAR domain as described herein. These and other types of single domain antibodies / ISVD are well known in the art and for example include a "dAb" (or an amino acid sequence that is suitable for use as a dAb) or a Nanobody™ (as defined herein, and including but not limited to a VHH sequence); other single variable domains, or any suitable fragment of any one thereof. For a general description of (single) domain antibodies, reference is also made to the publications cited above, as well as to EP 0368684. For the term “dAb’s”, reference is for example made to Ward et al. (Nature 1989 Oct 12; 341 (6242): 544-6), to Holt et al., Trends Biotechnol., 2003, 21(11):484-490; as well as to for example WO 06 / 030220, WO 06 / 003388 and other published patent applications of Domantis Ltd. It should also be noted that, although less preferred in the context of the present invention because they are not of mammalian origin, single domain antibodies or single variable domains can be derived from certain species of shark (for example, the so-called “IgNAR domains”, see for example WO 05 / 18629). For a description of VHHs and Nanobodies, reference is made to WO 08 / 020079 and the further references cited herein. Preferably, a single domain antibody, single variable domain or immunoglobulin single variable domains / ISVD as used in the invention will be a Nanobody (including VHHs and Nanobodies of synthetic or semi-synthetic origin, in each case carrying one or more of the “Hallmark residues” that are characteristic of Nanobodies, for which reference is again made to WO 08 / 020079).

[0108] An “antigen” is a molecule comprising at least one epitope. The antigen may for example be a polypeptide, nucleic acid, polysaccharide, protein, lipoprotein or glycoprotein.

[0109] A “complementarity determining region” or “CDR” is a hypervariable region of the antigen-binding region of an antibody. The CDRs are interspersed between regions that are more conserved, termed framework regions (FRs). The antigen-binding region of an antibody may thus comprise one or more CDRs and FRs, usually in each variable domain three CDRs and four FRsarranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0110] The terms “antigenic determinant” and “epitope”, which may also be used interchangeably herein. have the meanings given to it in paragraph l) on page 53 of WO 08 / 020079. In particular, the term “epitope” may refer to a determinant capable of specific binding to an antibody. Epitopes may for example be comprised within polypeptides or proteins. Epitopes may be continuous or discontinuous, wherein a discontinuous epitope is a conformational epitope on an antigen which is formed from at least two separate regions in the primary sequence of the protein, nucleic acid or polysaccharide.

[0111] As further described in paragraph m) on page 53 of WO 08 / 020079, an amino acid sequence (such as a Nanobody, an antibody, a polypeptide of the invention, or generally an antigen binding protein or polypeptide or a fragment thereof) that can (specifically) bind to, that has affinity for and / or that has specificity for a specific antigenic determinant, epitope, antigen or protein (or for at least one part, fragment or epitope thereof) is said to be “against” or “directed against” said antigenic determinant, epitope, antigen or protein.

[0112] The term “specificity” has the meaning given to it in paragraph n) on pages 53-56 of WO 08 / 020079; and as mentioned therein refers to the number of different types of antigens or antigenic determinants to which a particular antigen-binding molecule or antigen-binding protein (such as a Nanobody or a polypeptide of the invention) molecule can bind. The specificity of an antigen-binding protein can be determined based on affinity and / or avidity, as described on pages 53-56 of WO 08 / 020079 (incorporated herein by reference), which also describes some preferred techniques for measuring binding between an antigen-binding molecule (such as a Nanobody or polypeptide of the invention) and the pertinent antigen. Typically, antigen-binding proteins (such as the amino acid sequences, Nanobodies and / or polypeptides of the invention) will bind to their antigen with a dissociation constant (KD) of 10-5to 10-12moles / liter or less, and preferably 10-7to 10-12moles / liter or less and more preferably 10-8to 10-12moles / liter (i.e. with an association constant (KA) of 105to 1012liter / moles or more, and preferably 107to 1012liter / moles or more and more preferably 108to 1012liter / moles). Any KD value greater than 104mol / liter (or any KA value lower than 104M-1) liters / mol is generally considered to indicate non-specific binding. Preferably, a monovalent immunoglobulin sequence of the invention will bind to the desired antigen with an affinity less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 pM. Specific binding of an antigen-binding protein to an antigen or antigenic determinant can be determined in any suitable manner known per se, including, for example, Scatchard analysis and / or competitive binding assays, such as radioimmunoassays(RIA), enzyme immunoassays (EIA) and sandwich competition assays, and the different variants thereof known per se in the art; as well as the other techniques mentioned herein.

[0113] As will be clear to the skilled person, and as described on pages 53-56 of WO 08 / 020079, the dissociation constant may be the actual or apparent dissociation constant. Methods for determining the dissociation constant will be clear to the skilled person, and for example include the techniques mentioned on pages 53-56 of WO 08 / 020079.

[0114] As used herein, the term “affinity” refers to the strength of binding between receptors and their ligands, for example between an antibody and its antigen. The affinity of an antibody can be defined in terms of the dissociation constant, KD, which is an equilibrium constant that measures the propensity of a molecular complex to separate (dissociate) reversibly into the molecules forming the complex. In one aspect, KD is defined as the ratio koff / kon , where koff and kon are the rate constants for association and dissociation of the molecular complex. Preferably affinity is determined by calculating the dissociation constant KD based on IC50 values. Thus, the affinity is measured as an apparent affinity.

[0115] The term “residues”, as used herein, generally refers to single monomers collectively making one polymer. More specifically, the monomers combined in making the single-domain antibodies provided herein, consist of amino acids making up a polypeptide or protein.

[0116] The term “linker”, as used herein, refers to a molecular moiety used to covalently bind two molecules to one another. The linker may be of varying length and structure and may comprise different anchoring groups. In one embodiment, the anchoring group may be selected from the group consisting of amine, carboxylic acid, acid chloride, N-hydroxysuccinimide ester, maleimide, thiol, and a polypeptide.

[0117] The term “treatment”, as used herein, refers to any process, action, application, therapy, or the like, wherein a subject, including a human being, is subject to medical aid with the object of improving the animal’s condition, directly or indirectly. Thus, a treatment may involve curative treatment, ameliorating treatment and / or prophylactic treatment, where prophylactic treatment can result in complete prevention of a clinical and / or physiological condition or reduce the severity, such as reducing the number of and severity of any symptom associated with the clinical and / or physiological condition.

[0118] For the purposes of comparing two or more nucleotide sequences, the percentage of “sequence identity” between a first nucleotide sequence and a second nucleotide sequence may be calculated or determined as described in paragraph c) on page 49 of WO 08 / 020079 (incorporated herein by reference), such as by dividing [the number of nucleotides in the first nucleotide sequence that are identical to the nucleotides at the corresponding positions in the secondnucleotide sequence] by [the total number of nucleotides in the first nucleotide sequence] and multiplying by [100%], in which each deletion, insertion, substitution or addition of a nucleotide in the second nucleotide sequence - compared to the first nucleotide sequence - is considered as a difference at a single nucleotide (position); or using a suitable computer algorithm or technique, again as described in paragraph c) on pages 49 of WO 08 / 020079 (incorporated herein by reference).

[0119] For the purposes of comparing two or more amino acid sequences, the percentage of “sequence identity” between a first amino acid sequence and a second amino acid sequence (also referred to herein as “amino acid identity”) may be calculated or determined as described in paragraph f) on pages 49 and 50 of WO 08 / 020079 (incorporated herein by reference), such as by dividing [the number of amino acid residues in the first amino acid sequence that are identical to the amino acid residues at the corresponding positions in the second amino acid sequence] by [the total number of amino acid residues in the first amino acid sequence] and multiplying by [100%], in which each deletion, insertion, substitution or addition of an amino acid residue in the second amino acid sequence - compared to the first amino acid sequence - is considered as a difference at a single amino acid residue (position), i.e. as an “amino acid difference” as defined herein; or using a suitable computer algorithm or technique, again as described in paragraph f) on pages 49 and 50 of WO 08 / 020079 (incorporated herein by reference).

[0120] Also, in determining the degree of sequence identity between two amino acid sequences, the skilled person may take into account so-called “conservative” amino acid substitutions, as described on page 50 of WO 08 / 020079.

[0121] Any amino acid substitutions applied to the polypeptides described herein may also be based on the analysis of the frequencies of amino acid variations between homologous proteins of different species developed by Schulz et al., Principles of Protein Structure, Springer-Verlag, 1978, on the analyses of structure forming potentials developed by Chou and Fasman, Biochemistry 13: 211, 1974 and Adv. Enzymol., 47: 45-149, 1978, and on the analysis of hydrophobicity patterns in proteins developed by Eisenberg et al., Proc. Nad. Acad Sci. USA 81: 140-144, 1984; Kyte & Doolittle; J Molec. Biol. 157: 105-132, 1981, and Goldman et al., Ann. Rev. Biophys. Chem. 15: 321-353, 1986, all incorporated herein in their entirety by reference. Information on the primary, secondary and tertiary structure of Nanobodies is given in the description herein and in the general background art cited above. Also, for this purpose, the crystal structure of a VHH domain from a llama is for example given by Desmyter et al., Nature Structural Biology, Vol.3, 9, 803 (1996); Spinelli et al., Natural Structural Biology (1996); 3, 752-757; and Decanniere et al., Structure, Vol. 7, 4, 361 (1999). Further information about some of the aminoacid residues that in conventional VH domains form the VH / VL interface and potential camelizing substitutions on these positions can be found in the publications cited above.

[0122] Methods for determining amino acid sequence “identity” are known in the art. By way of example, a polypeptide of interest may comprise an amino acid sequence having at least 70%, 80%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity with the amino acid sequence of a reference polypeptide. There are many established algorithms available to align two amino acid sequences. Typically, one sequence acts as a reference sequence, to which test sequences may be compared. The sequence comparison algorithm calculates the percentage sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters. Alignment of amino acid sequences for comparison may be conducted, for example, by computer implemented algorithms (e.g., GAP, BESTFIT, FASTA or TFASTA), or BLAST and BLAST 2.0 algorithms. The BLOSUM62 table shown below is an amino acid substitution matrix derived from about 2,000 local multiple alignments of protein sequence segments, representing highly conserved regions of more than 500 groups of related proteins (Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-10919, 1992; incorporated herein by reference). Amino acids are indicated by the standard one-letter codes. The percent identity is calculated as: Total number of identical matches __________________________________________ x 100 [length of the longer sequence plus the number of gaps Introduced into the longer sequence in order to align the two sequences] BLOSUM62 table A R N D C Q E G H I L K M F P S T W Y V A 4 R -1 5 N -2 0 6 D -2 -2 1 6 C 0 -3 -3 -3 9 Q -1 1 0 0 -3 5 E -1 0 0 2 -4 2 5 G 0 -2 0 -1 -3 -2 -2 6 H -2 0 1 -1 -3 0 0 -2 8I -1 -3 -3 -3 -1 -3 -3 -4 -3 4 L -1 -2 -3 -4 -1 -2 -3 -4 -3 2 4 K -1 2 0 -1 -3 1 1 -2 -1 -3 -2 5 M -1 -1 -2 -3 -1 0 -2 -3 -2 1 2 -1 5 F -2 -3 -3 -3 -2 -3 -3 -3 -1 0 0 -3 0 6 P -1 -2 -2 -1 -3 -1 -1 -2 -2 -3 -3 -1 -2 -4 7 S 1 -1 1 0 -1 0 0 0 -1 -2 -2 0 -1 -2 -1 4 T 0 -1 0 -1 -1 -1 -1 -2 -2 -1 -1 -1 -1 -2 -1 1 5 W -3 -3 -4 -4 -2 -2 -3 -2 -2 -3 -2 -3 -1 1 -4 -3 -211 Y -2 -2 -2 -3 -2 -1 -2 -3 2 -1 -1 -2 -1 3 -3 -2 -2 2 7 V 0 -3 -3 -3 -1 -2 -2 -3 -3 3 1 -2 1 -1 -2 -2 0 -3 -1 4

[0123] Sequence identity may exist over a region of the sequences that is at least e.g., 10 amino acid residues in length (e.g., at least 15, 20, 30, 40, 50, 75, 100 amino acid residues in length) – e.g., up to the entire length of the reference sequence. Substantially homologous polypeptides have one or more amino acid substitutions, deletions, or additions. In many embodiments, those changes are of a minor nature, for example, involving only conservative amino acid substitutions. Conservative substitutions are those made by replacing one amino acid with another amino acid within the following groups: Basic: arginine, lysine, histidine; Acidic: glutamic acid, aspartic acid; Polar: glutamine, asparagine; Hydrophobic: leucine, isoleucine, valine; Aromatic: phenylalanine, tryptophan, tyrosine; Small: glycine, alanine, serine, threonine, methionine. Substantially homologous polypeptides also encompass those comprising other substitutions that do not significantly affect the folding or activity of the polypeptide; small deletions, typically of 1 to about 30 amino acids (such as 1-10, or 1-5 amino acids); and small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue, a small linker peptide of up to about 20-25 residues, or an affinity tag.

[0124] Amino acid sequences and nucleic acid sequences are said to be “exactly the same” if they have 100% sequence identity (as defined herein) over their entire length;

[0125] When comparing two amino acid sequences, the term “amino acid difference” refers to an insertion, deletion or substitution of a single amino acid residue on a position of the first sequence, compared to the second sequence; it being understood that two amino acid sequences can contain one, two or more such amino acid differences;

[0126] The term “humanization” (and corresponding terms used herein such as "humanized") has its usual meaning in the art and generally refers to replacing one or more amino acid residues inthe amino acid sequence of a naturally occurring antibody sequence (and in particular in the framework sequences thereof) that has been derived from an animal (and in particular a mammal) other than a human being with one or more of the amino acid residues that occur at the corresponding position(s) in the amino acid sequence of an antibody sequence that has been derived from a human being. This can be performed in a manner known per se, which will be clear to the skilled person, for example on the basis of the further description herein and the publications on humanization referred to herein. For example and without limitation, for the humanization of VHH and Nanobodies, the tables on pages 79 to 82 mention, for the different positions in the amino acid sequence of a VHH or Nanobody, some of the amino acid residues that are present at the corresponding position in a human VH3 sequence (as well as the so-called “Hallmark residues” that are commonly found in the amino acid sequence of Nanobodies). It should also be noted that, in the practice of the invention, C1q binders of the invention can be generated by first (suitably) introducing, into the sequence of a naturally occurring C1q binder, one or more mutations that reduce the affinity of the C1q binder for C1q (as further described herein) and then (suitably) introducing, into the variant of the naturally occurring C1q binder thus obtained, one or more humanized substitutions; or alternatively C1q binders of the invention can be generated by first (suitably) introducing, into the sequence of a naturally occurring C1q binder, one or more humanizing substitutions, and suitably) introducing, into the sequence of a said humanized variant, one or more mutations that reduce the affinity of the C1q binder for C1q (as further described herein); or any suitable combination of the foregoing. Is should also be noted that, in particular with respect to any antibody or antibody-derived sequence, the term “naturally occurring” generally relates to a sequence that is or can be generated from the immune repertoire of a suitable species of animal (and in particular a suitable species of mammal, such as – in the case of a naturally occurring VHH sequence -a species of Camelid (including transgenic animals from other mammalian species other than Camelids whose immune repertoire has been “camelized”). Methods of generating such naturally occurring (antibody) sequences will be clear to the skilled person, and for example include screening, with the antigen of interest, a suitable immune library (in particular an such immune library that has been obtained from an animal that has been suitably immunized with the antigen of interest) or for example screening, with the antigen of interest, of an immune library that has been obtained from an animal (and in particular a species of mammal, such as a species of Camelid), optionally followed by affinity maturation or similar techniques.

[0127] When a nucleotide sequence or amino acid sequence is said to “comprise” another nucleotide sequence or amino acid sequence, respectively, or to “essentially consist of” anothernucleotide sequence or amino acid sequence, this has the meaning given in paragraph i) on pages 51-52 of WO 08 / 020079.

[0128] The term “in essentially isolated form” has the meaning given to it in paragraph j) on pages 52 and 53 of WO 08 / 020079.

[0129] The terms “domain” and “binding domain” have the meanings given to it in paragraph k) on page 53 of WO 08 / 020079.

[0130] The half-life of an amino acid sequence, compound or polypeptide of the invention can generally be defined as described in paragraph o) on page 57 of WO 08 / 020079 and as mentioned therein refers to the time taken for the serum concentration of the amino acid sequence, compound or polypeptide to be reduced by 50%, in vivo, for example due to degradation of the sequence or compound and / or clearance or sequestration of the sequence or compound by natural mechanisms. The in vivo half-life of an amino acid sequence, compound or polypeptide of the invention can be determined in any manner known per se, such as by pharmacokinetic analysis. Suitable techniques will be clear to the person skilled in the art and may for example generally be as described in paragraph o) on page 57 of WO 08 / 020079. As also mentioned in paragraph o) on page 57 of WO 08 / 020079, the half-life can be expressed using parameters such as the t1 / 2-alpha, t1 / 2-beta and the area under the curve (AUC). Reference is for example made to the Experimental Part below, as well as to the standard handbooks, such as Kenneth, A et al: Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists and Peters et al, Pharmacokinetic analysis: A Practical Approach (1996). Reference is also made to "Pharmacokinetics", M Gibaldi & D Perron, published by Marcel Dekker, 2nd Rev. edition (1982). The terms “increase in half-life” or “increased half-life” as also as defined in paragraph o) on page 57 of WO 08 / 020079 and in particular, refer to an increase in the t1 / 2-beta, either with or without an increase in the t1 / 2-alpha and / or the AUC or both.

[0131] In the context of the present invention, “modulating” or “to modulate” generally means either reducing or inhibiting the activity of, or alternatively increasing the activity of, a target or antigen, as measured using a suitable in vitro, cellular or in vivo assay. In particular, “modulating” or “to modulate” may mean either reducing or inhibiting the activity of, or alternatively increasing a (relevant or intended) biological activity of, a target or antigen, as measured using a suitable in vitro, cellular or in vivo assay (which will usually depend on the target or antigen involved), by at least 1%, preferably at least 5%, such as at least 10% or at least 25%, for example by at least 50%, at least 60%, at least 70%, at least 80%, or 90% or more, compared to activity of the target or antigen in the same assay under the same conditions but without the presence of the construct of the invention.

[0132] As will be clear to the skilled person, “modulating” may also involve effecting a change (which may either be an increase or a decrease) in affinity, avidity, specificity and / or selectivity of a target or antigen for one or more of its ligands, binding partners, partners for association into a homomultimeric or heteromultimeric form, or substrates; and / or effecting a change (which may either be an increase or a decrease) in the sensitivity of the target or antigen for one or more conditions in the medium or surroundings in which the target or antigen is present (such as pH, ion strength, the presence of co-factors, etc.), compared to the same conditions but without the presence of the construct of the invention. As will be clear to the skilled person, this may again be determined in any suitable manner and / or using any suitable assay known per se, depending on the target or antigen involved.

[0133] “Modulating” may also mean effecting a change (i.e. an activity as an agonist, as an antagonist or as a reverse agonist, respectively, depending on the target or antigen and the desired biological or physiological effect) with respect to one or more biological or physiological mechanisms, effects, responses, functions, pathways or activities in which the target or antigen (or in which its substrate(s), ligand(s) or pathway(s) are involved, such as its signalling pathway or metabolic pathway and their associated biological or physiological effects) is involved. Again, as will be clear to the skilled person, such an action as an agonist or an antagonist may be determined in any suitable manner and / or using any suitable (in vitro and usually cellular or in assay) assay known per se, depending on the target or antigen involved. In particular, an action as an agonist or antagonist may be such that an intended biological or physiological activity is increased or decreased, respectively, by at least 1%, preferably at least 5%, such as at least 10% or at least 25%, for example by at least 50%, at least 60%, at least 70%, at least 80%, or 90% or more, compared to the biological or physiological activity in the same assay under the same conditions but without the presence of the construct of the invention.

[0134] Modulating may for example also involve allosteric modulation of the target or antigen; and / or reducing or inhibiting the binding of the target or antigen to one of its substrates or ligands and / or competing with a natural ligand, substrate for binding to the target or antigen. Modulating may also involve activating the target or antigen or the mechanism or pathway in which it is involved. Modulating may for example also involve effecting a change in respect of the folding or confirmation of the target or antigen, or in respect of the ability of the target or antigen to fold, to change its confirmation (for example, upon binding of a ligand), to associate with other (sub)units, or to disassociate. Modulating may for example also involve effecting a change in the ability of the target or antigen to transport other compounds or to serve as a channel for other compounds(such as ions). Modulating may be reversible or irreversible, but for pharmaceutical and pharmacological purposes will usually be in a reversible manner.

[0135] In respect of a target or antigen, the term “interaction site” on the target or antigen means a site, epitope, antigenic determinant, part, domain or stretch of amino acid residues on the target or antigen that is a site for binding to a ligand, receptor or other binding partner, a catalytic site, a cleavage site, a site for allosteric interaction, a site involved in multimerisation (such as homomerization or heterodimerization) of the target or antigen; or any other site, epitope, antigenic determinant, part, domain or stretch of amino acid residues on the target or antigen that is involved in a biological action or mechanism of the target or antigen. More generally, an “interaction site” can be any site, epitope, antigenic determinant, part, domain or stretch of amino acid residues on the target or antigen to which an amino acid sequence or polypeptide of the invention can bind such that the target or antigen (and / or any pathway, interaction, signalling, biological mechanism or biological effect in which the target or antigen is involved) is modulated (as defined herein).

[0136] An amino acid sequence or polypeptide is said to be “specific for” a first target or antigen compared to a second target or antigen when is binds to the first antigen with an affinity (as described above, and suitably expressed as a KD value, KA value, Koff rate and / or Kon rate) that is at least 10 times, such as at least 100 times, and preferably at least 1000 times, and up to 10.000 times or more better than the affinity with which said amino acid sequence or polypeptide binds to the second target or polypeptide. For example, the first antigen may bind to the target or antigen with a KD value that is at least 10 times less, such as at least 100 times less, and preferably at least 1000 times less, such as 10.000 times less or even less than that, than the KDwith which said amino acid sequence or polypeptide binds to the second target or polypeptide. Preferably, when an amino acid sequence or polypeptide is “specific for” a first target or antigen compared to a second target or antigen, it is directed against (as defined herein) said first target or antigen, but not directed against said second target or antigen.

[0137] The terms “cross-block”, “cross-blocked” and “cross-blocking” are used interchangeably herein to mean the ability of an amino acid sequence or other binding agents (such as a polypeptide of the invention) to interfere with the binding of other amino acid sequences or binding agents of the invention to a given target. The extent to which an amino acid sequence or other binding agents of the invention interfere with the binding of another to a target described herein can be determined using competition binding assays. One particularly suitable quantitative assay uses a Biacore machine which can measure the extent of interactions using surface plasmon resonance technology. Another suitable quantitative cross-blocking assay uses an ELISA-based approach to measure competition between amino acid sequence or another binding agent in terms of theirbinding to the target. Also, as will be clear to the skilled person, two binding domains, binding units or antibodies that are directed against the same binding site or epitope on an antigen (and / or that are described herein as being directed against the same binding site or epitope) will compete for binding to said binding site or epitope (as determined in a suitable competitive binding assay) and / or will able to cross-block each other’s binding to said binding site or epitope.

[0138] As further described herein, the total number of amino acid residues in a Nanobody can be in the region of 110-120, is preferably 112-115, and is most preferably 113. It should however be noted that parts, fragments, analogs or derivatives (as further described herein) of a Nanobody are not particularly limited as to their length and / or size, as long as such parts, fragments, analogs or derivatives meet the further requirements outlined herein and are also preferably suitable for the purposes described herein;

[0139] The amino acid residues of a Nanobody are numbered according to the general numbering for VH domains given by Kabat et al. ("Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, MD, Publication No.91), as applied to VHH domains from Camelids in the article of Riechmann and Muyldermans, Journal of Immunological Methods 231 199925–38 (see for example Figure 2 of said article or referred to herein. Reference is also made to the following website: http: / / www.bioinf.org.uk / abs / info.html#kabatnum). According to this numbering, FRl of a Nanobody comprises the amino acid residues at positions 1-30, CDRl of a Nanobody comprises the amino acid residues at positions 31-35, FR2 of a Nanobody comprises the amino acids at positions 36-49, CDR2 of a Nanobody comprises the amino acid residues at positions 50-65, FR3 of a Nanobody comprises the amino acid residues at positions 66-94, CDR3 of a Nanobody comprises the amino acid residues at positions 95-102, and FR4 of a Nanobody comprises the amino acid residues at positions 103-113. [In this respect, it should be noted that - as is well known in the art for VH domains and for VHH domains - the total number of amino acid residues in each of the CDR' s may vary and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (that is, one or more positions according to the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed for by the Kabat numbering). This means that, generally, the numbering according to Kabat may or may not correspond to the actual numbering of the amino acid residues in the actual sequence. Generally, however, it can be said that, according to the numbering of Kabat and irrespective of the number of amino acid residues in the CDR' s, position 1 according to the Kabat numbering corresponds to the start of FRl and vice versa, position 36 according to the Kabat numbering corresponds to the start of FR2 and vice versa, position 66 according to the Kabat numbering corresponds to the start of FR3 and vice versa, and position 103according to the Kabat numbering corresponds to the start of FR4 and vice versa.]. Alternative methods for numbering the amino acid residues of VH domains, which methods can also be applied in an analogous manner to VHH domains from Camelids and to Nanobodies, are the method described by Chothia et al. (Nature 342, 877-883 (1989)), the so-called "AbM definition" and the so-called "contact definition". However, in the present description, claims and figures, the numbering according to Kabat as applied to VHH domains by Riechmann and Muyldermans will be followed, unless indicated otherwise.

[0140] It should be noted that some of the mutations disclosed herein are (also) described with reference to their position in the sequence of the preferred C1 binder in which they can be made (in particular, in the sequence of Nb75 or Nb78, respectively. If so, these mutations may also be mentioned herein with reference to their position in the sequence of Nb75 or Nb78, respectively, rather than according to the Kabat numbering. For the sake of reference, Table E below is a transposition Table listing: (i) the amino acid residues in the CDRs of Nb75 and Nb78, respectively (in bold / double underline; see column II, V and VII for Nb78 and column III, VI and IX for Nb 75, respectively); (ii) the position according to the Kabat numbering (columns I, IV and VII); and (iii) for CDR3, the numbering used when reference is made to the sequences for Nb78 and 78. Also for the sake of reference, Table E mentions some of the amino acid residues that are present in the framework regions that flank the CDRs. Thus, by means of example and as will be clear to the skilled person from Table E, the M33A mutation mentioned herein for CDR1 of Nb78 will be at position 52 according to Kabat; the T53G mutation mentioned herein for CDR2 of Nb75 will be at position 52 according to Kabat; the T102A mutation mentioned herein for CDR3 of Nb78 will be at position 96 according to Kabat; and the S103A mutation mentioned herein for CDR3 of Nb78 will be at position 97 according to Kabat. Also, based on the disclosure herein with respect to mutation(s) for Nb75 and Nb78, the skilled person will be able to make and test similar or equivalent mutations in other C1q binders, for example in some of the other C1q binders mentioned herein.

[0141] With reference to a C1q binder, the wording “with reduced affinity for C1q” is generally used herein to indicate that said C1q binder has a binding affinity to the C1q complement factor of from about 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 particular, such a C1q binder may bind to the C1q complement factor with a KDfrom 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. As also described in more detail herein, a C1q binder “with reduced affinity for C1q” may be a variant ofa naturally occurring (i.e., “wild-type”) C1q binder that comprises one or more modifications relative to said wild-type C1q binder, with said one or more modifications resulting in reduced binding affinity to a C1q complement factor as compared to a binding affinity of the corresponding wild-type C1q binder. For example and without limitation, in one particular aspect, such a C1q binder “with reduced affinity for C1q” may be a variant of Nb75 with reduced affinity for C1q compared to Nb75 or a variant of Nb78 with reduced affinity for C1q compared to Nb78 (reference is again made to the further description herein). Also, in the present description and claims, the term: “affinity for C1q” in particular refers to affinity for the globular head of C1q, and more in particular affinity for the globular head of C1q as determined by the method set out in Example 1.

[0142] The term “C1q binder” (which term is used interchangeably herein with the wording “complement factor-engaging domain that binds to C1q”) refers to a complement factor-engaging domain that binds to C1q, which complement factor-engaging domain is as further described herein. As further described herein, according to one preferred but non-limiting aspect, such a C1q binder is a C1q binder “with reduced affinity for C1q” as further described herein. As also described herein, according to a particular aspect of the invention, a C1q binder that is used in the invention is preferably a single domain antibody (also sometimes referred to as an immunoglobulin single variable domain), and more preferably a Nanobody (including VHHs obtained from a Camelid as well as for example Nanobodies from synthetic or semi-synthetic origin).

[0143] Unless explicitly indicated otherwise herein, when a C1q binder is said to be “linked or fused to” an antibody heavy chain or light chain, it may be linked or fused to such heavy or light chain in any suitable manner (i.e., at a position and in a manner that essentially does not interfere with the desired action(s) of said C1q binder and heavy or light chain). In particular, and unless explicitly indicated otherwise herein, when a C1q binder is said to be “linked or fused to” an antibody heavy chain or light chain, it may be linked or fused to the N-terminus or to the C- terminus of such heavy chain or light chain. According to a specific but non-limiting aspect, examples of which are given in the Experimental Part below, when a C1q binder is said to be “linked or fused to” an antibody heavy chain, it may in practice in particular be linked or fused to the N-terminus of such heavy chain; and when a C1q binder is said to be “linked or fused to” an antibody light heavy chain, it may in practice in particular be linked or fused to the C-terminus of such light chain.

[0144] Also, when two C1q binders are said to be “linked or fused to” an “antibody of the invention” (as described herein), each such C1q binder may be the same or different, but they are preferably the same. Furthermore, when two C1q binders are said to be linked or fused to an “antibody of the invention” (as described herein), each of said C1q binders may, independentlyfrom the other, be linked or fused (as described herein) to the heavy chain or to the light chain; but in the practice of the invention, and preferably, either both C1q binders will be linked or fused to the two heavy chains (with one C1q binder being linked or fused to one of the heavy chains and the other being linked or fused to the other heavy chain) or alternatively both C1q binders will be linked or fused to the two light chains (with one C1q binder being linked or fused to one of the light chains and the other being linked or fused to the other light chain). Also, in the practice of the invention, and preferably, when one of the C1q binders is linked or fused to the C-terminus of a heavy or light chain, respectively, that is present in an antibody of the invention, the other C1q binder will be linked or fused to the C-terminus of other heavy or light chain, respectively, that is present in said antibody of the invention; and conversely; when one of the C1q binders is linked or fused to the N-terminus of a heavy or light chain, respectively, that is present in an antibody of the invention, the other C1q binder will be linked or fused to the N-terminus of other heavy or light chain, respectively, that is present in said antibody of the invention. According to a specific but non-limiting aspect, examples of which are given in the Experimental Part below, when an antibody of the invention contains two C1q binders that are each linked or fused to one of the heavy chains that are present in said antibody of the invention, one of said C1q binders may in particular be linked or fused to the N-terminus of one of the heavy chains, and the other C1q binder may be linked or fused to the N-terminus of the other heavy chain; or alternatively, when an antibody of the invention contains two C1q binders that are each linked or fused to one of the light chains that are present in said antibody of the invention, one of said C1q binders may in particular be linked or fused to the C-terminus of one of the light chains, and the other C1q binder may be linked or fused to the C-terminus of the other light chain.

[0145] The term “antibody of the invention” generally refers to an antibody comprising two antibody heavy chains (with each heavy chain comprising a VH domain, a CH1 domain and an FC portion, with the Fc portion being comprised of a CH2 domain and a CH3 domain) and two antibody light chains (with each light chain comprising a VL and a CL domain), which antibody suitably comprises at least one C1q binder as further described herein (i.e. a C1q binder having with reduced affinity for C1q) that is “linked or fused to” (as described herein) said antibody (i.e. to one of the heavy chains or to one of the light chains). As further described herein, usually and preferably, such an antibody of the invention will suitably comprise two such C1q binders that are linked or fused to (as described herein) said antibody.

[0146] The Figures, Sequence Listing and the Experimental Part / Examples are only given to further illustrate the invention and should not be interpreted or construed as limiting the scope ofthe invention and / or of the appended claims in any way, unless explicitly indicated otherwise herein.

[0147] For a general description of heavy chain antibodies and the variable domains thereof, reference is inter alia made to the publications cited herein, as well as to the publications mentioned on page 59 of WO 08 / 020079 and to the list of references mentioned on pages 41-43 of the International Application WO 06 / 040153, which publications and references are incorporated herein by reference. For example, pages 72 to 82 of WO 08 / 020079 give a detailed description of some of the characteristics (including the so-called “Hallmark residues”) that are commonly found in the amino acid sequence of Nanobodies.

[0148] In accordance with the terminology used in the art (see the above references), the variable domains present in naturally occurring heavy chain antibodies will also be referred to as “VHH domains”, in order to distinguish them from the heavy chain variable domains that are present in conventional 4-chain antibodies (which will be referred to hereinbelow as “VH domains”) and from the light chain variable domains that are present in conventional 4-chain antibodies (which will be referred to hereinbelow as “VL domains”).

[0149] As mentioned in the publications referred to above, VHH domains have a number of unique structural characteristics and functional properties which make isolated VHHdomains (as well as Nanobodies based thereon, which share these structural characteristics and functional properties with the naturally occurring VHHdomains) and proteins containing the same highly advantageous for use as functional antigen-binding domains or proteins. In particular, and without being limited thereto, VHHdomains (which have been “designed” by nature to functionally bind to an antigen without the presence of, and without any interaction with, a light chain variable domain) and Nanobodies can function as a single, relatively small, functional antigen-binding structural unit, domain or protein. This distinguishes the VHH domains from the VH and VL domains of conventional 4-chain antibodies, which by themselves are generally not suited for practical application as single antigen-binding proteins or domains, but need to be combined in some form or another to provide a functional antigen-binding unit (as in for example conventional antibody fragments such as Fab fragments; in ScFv’s fragments, which consist of a VH domain covalently linked to a VLdomain).

[0150] Because of these unique properties, the use of VHH domains and Nanobodies as single antigen-binding proteins or as antigen-binding domains (i.e. as part of a larger protein or polypeptide) offers a number of significant advantages over the use of conventional VH and VL domains, scFv’s or conventional antibody fragments (such as Fab- or F(ab’)2-fragments), including the advantages that are listed on pages 60 and 61 of WO 08 / 020079.Overview

[0151] Disclosed herein are complement factor-engaging polypeptides and complement factor- engaging domains that bind to a C1q complement factor with optimized affinity (for example, low affinity). In some aspects, the complement factor-engaging polypeptides and complement factor- engaging domains that bind to the C1q complement factor with optimized affinity can be linked to an antigen-binding moiety that binds to a target associated with a disease or condition. Such bispecific / multi-specific constructs as disclosed herein can have a higher affinity for the target associated with the disease or condition than the affinity for C1q complement factor. Consequently, the constructs as disclosed herein can comprise a low affinity complement factor- engaging polypeptide or complement factor-engaging domain that binds to and modulates the activity of the complement system of a subject to treat a disease or condition described herein. By engaging with C1q through the low affinity complement factor-engaging polypeptide / domain and the target associated with the disease or condition, the construct is able to recruit the complement system to the site of the target associated with the disease or condition, thus inducing a targeted immune response at the site of the target. Provided herein are bispecific constructs comprising a complement factor-engaging domain that binds to a complement factor with a binding affinity of about 1 nM to about 10 μM, as determined by biolayer interferometry, directly or indirectly linked to an antigen-binding moiety that binds to a target antigen such as a target protein.

[0152] In some embodiments, the complement factor-engaging polypeptides or domains of the present disclosure are single domain antibodies. In some embodiments, the complement factor- engaging domains of the present disclosure are provided in a bispecific construct also comprising an antigen-binding moiety that binds to a target antigen. Without wishing to be bound by theory, the complement factor-engaging polypeptides or domains described herein which are single domain antibodies have greater flexibility, and thus more robust engagement of complement and the target associated with the disease or condition, as compared to a bispecific antibody that utilize a full-length complement factor-engaging antibody. Further, the present application demonstrates that bispecific constructs having a low affinity C1q single domain antibody coupled to a high affinity single domain antibody directed against the second target surprisingly induce greater complement-mediated activity at the site of the second target, as compared to constructs with a high affinity C1q single domain antibody coupled to a high affinity single domain antibody directed against the second target.

[0153] Accordingly, the bispecific constructs of the present disclosure can be used to treat diseases or conditions such as cancer, where the low affinity complement factor-engaging domain directedto C1q is linked to an antigen binding moiety directed against a target antigen, such as a target protein, associated with the disease or condition, such as cancer, resulting in superior complement- mediated cell cytotoxicity. Optimized C1q Complement Factor-Engaging Domain or Polypeptide

[0154] Disclosed herein are complement factor-engaging domains that bind to a C1q complement factor with optimized affinity (for example, low affinity). Such optimized C1q complement factor- engaging domains can be included in a bispecific construct as described herein. A complement factor-engaging domain or C1q complement factor-engaging domain as described herein may be such an optimized C1q complement factor-engaging domain.

[0155] Also disclosed herein are C1q complement factor-engaging polypeptides such as engineered C1q complement factor-engaging polypeptides. A C1q complement factor-engaging polypeptide may comprise or may consist of a complement factor-engaging domain as described herein. A C1q complement factor-engaging polypeptide may comprise or may consist of a C1q complement factor-engaging domain as described herein. A C1q complement factor-engaging polypeptide may comprise or may consist of an optimized C1q complement factor-engaging domain as described herein.

[0156] In some embodiments, the complement factor-engaging domain comprises a single chain antibody, such as a single chain antibody as described herein. In some embodiments, the complement factor-engaging domain comprises a heavy chain constant region (CH), a heavy chain variable region (VH), a light chain constant region (CL), a light chain variable region (VL), or fragment of any thereof, of an antibody. In some embodiments, the complement factor-engaging domain comprises a VHH domain. In some embodiments, the complement factor-engaging domain comprises a VNAR domain. In some embodiments, the complement factor-engaging domain comprises a DARPin (designed ankyrin repeat protein) polypeptide. In some embodiments, the complement factor-engaging domain comprises is a KNOB domain peptide.

[0157] In some cases, the C1q complement factor-engaging domain has a sequence at least 70%, 80%, 90%, 95%, 97%, 99% sequence identity to any one of SEQ ID NO: 1 – SEQ ID NO: 42 (TABLE 1). In some embodiments, the C1q complement factor-engaging domain has about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, about 99% sequence identity to any one of SEQ ID NO: 1 – SEQ ID NO: 42. In some embodiments, the complement factor-engaging domain has an amino acid sequence selected from SEQ ID Nos 4- 42. In some embodiments, the complement factor-engaging domain has an amino acid sequence comprising the amino acid sequence of any one of SEQ ID Nos 4-42. In some embodiments, thecomplement factor-engaging domain does not have a wild-type complement factor-engaging domain sequence, such as the amino acid sequence of any of SEQ ID NO: 1- SEQ ID NO: 3. In some embodiments, the complement factor-engaging domain does not comprise the amino acid sequence of SEQ ID NO: 1. In some embodiments, the complement factor-engaging domain does not comprise the amino acid sequence of SEQ ID NO: 2. In some embodiments, the complement factor-engaging domain does not comprise the amino acid sequence of SEQ ID NO: 3. TABLE 1: Exemplary C1q complement factor-engaging domains SEQ ID NO: SEQUENCE SEQ ID NO: 1 QVQLVETGGGLVQAGGSLRLSCAASGRTFNNDVMAWFRQAPGTERE FVALITAGGGTHYADSVKGRFVISRDNDKNMAYLQMNSLKSEDTAIY YCGADENPPGWPSRWSSAYDYWGQGTQVTVSSHHHHHH SEQ ID NO: 2 QVQLVESGGGLVQDGDSLRLSCAGSGWTFRDSMYNMGWFRQAPGK EREFVAAISWRGGSTLYADSVKGRFTISRDNAKNTVYLQMNSLKPEDT AVYQCAADTSARAALYSTGYEYDHWGQGTQVTVSS SEQ ID NO: 3 QVQLVETGGGLVQAGGSLRLSCAASGRTFNNDVMAWFRQAPGTERE FVALITAGGGTHYADSVKGRFVISRDNDKNMAYLQMNSLKSEDTAIY YCGADENPPGWPSRWSSAYDYWGQGTQVTVSS SEQ ID NO: 4 QVQLVESGGGLVQDGDSLRLSCAGSGWTFRDSMYNMGWFRQAPGK EREFVAAISWRGGSTLYADSVKGRFTISRDNAKNTVYLQMNSLKPEDT AVYQCAADTSARAALYSTGAEYDHWGQGTQVTVSS SEQ ID NO: 5 QVQLVESGGGLVQDGDSLRLSCAGSGWTFRDSMYNMGWFRQAPGK EREFVAAISWRGGSTLYADSVKGRFTISRDNAKNTVYLQMNSLKPEDT AVYQCAADTSARAALYSTGYAYDHWGQGTQVTVSS SEQ ID NO: 6 QVQLVESGGGLVQDGDSLRLSCAGSGWTFRDSMYNMGWFRQAPGK EREFVAAISWRGGSTLYADSVKGRFTISRDNAKNTVYLQMNSLKPEDT AVYQCAAATSARAALYSTGYEYDHWGQGTQVTVSS SEQ ID NO: 7 QVQLVESGGGLVQDGDSLRLSCAGSGWTFRDSMYNMGWFRQAPGK EREFVAAISWRGGSTLYADSVKGRFTISRDNAKNTVYLQMNSLKPEDT AVYQCAADASARAALYSTGAEYDHWGQGTQVTVSS SEQ ID NO: 8 QVQLVESGGGLVQDGDSLRLSCAGSGWTFRDSMYNMGWFRQAPGK EREFVAAISWRGGSTLYADSVKGRFTISRDNAKNTVYLQMNSLKPEDT AVYQCAADASARAALYSTGYAYDHWGQGTQVTVSS SEQ ID NO: 9 EVQLVESGGGLVQPGGSLRLSCAASGWTFRDSMYNMGWFRQAPGKG RELVAAISWRGGSTYYPDSVEGRFTISRDNAKRMVYLQMNSLRAEDT AVYYCAADTSARAALYSTGYEYDHWGQGTQVTVSS SEQ ID NO: 10 QVQLVESGGGSVQPGGSLRLSCTASGWTFRDSMYNLGWFRQAPGQE REAVAAISWRGGSTYYADSVKGRFTISRDNAKNTVTLQMNNLKPEDT AIYYCAADTSARAALYSTGYEYDHWGQGTQVTVSS SEQ ID NO: 11 QVQLVESGGGLVQPGGSLRLSCAASGWTFRDSMYNLGWFRQAPGQG LEAVAAISWRGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDT AVYYCAADTSARAALYSTGYEYDHWGQGTLVTVSS SEQ ID NO: 12 EVQLQASGGGFVQPGGSLRLSCAASGWTFRDSMYNMGWFRQAPGKE REFVSAISWRGGSTYYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTA TYYCAADTSARAALYSTGYEYDHWGQGTQVTVSSSEQ ID NO: 13 QVQLVESGGGLVQPGGSLRLSCAASGWTFRDSMYNLGWFRQAPGQG LEAVAAISWRGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDT AVYYCAADTSARAALYSTGYEYDHWGQGTLVTVSS SEQ ID NO: 14 QVQLVESGGGSVQPGGSLRLSCTASGWTFRASAYNLGWFRQAPGQER EAVAAISWRGGSTYYADSVKGRFTISRDNAKNTVTLQMNNLKPEDTA IYYCAADTSARAALYSTGYEYDHWGQGTQVTVSS SEQ ID NO: 15 QVQLVESGGGSVQPGGSLRLSCTASGWTFRDAAYNLGWFRQAPGQE REAVAAISWRGGSTYYADSVKGRFTISRDNAKNTVTLQMNNLKPEDT AIYYCAADTSARAALYSTGYEYDHWGQGTQVTVSS SEQ ID NO: 16 QVQLVESGGGSVQPGGSLRLSCTASGWTFRDTAYNLGWFRQAPGQER EAVAAISWRGGSTYYADSVKGRFTISRDNAKNTVTLQMNNLKPEDTA IYYCAADTSARAALYSTGYEYDHWGQGTQVTVSS SEQ ID NO: 17 QVQLVESGGGSVQPGGSLRLSCTASGWTFRQSAYNLGWFRQAPGQER EAVAAISWRGGSTYYADSVKGRFTISRDNAKNTVTLQMNNLKPEDTA IYYCAADTSARAALYSTGYEYDHWGQGTQVTVSS SEQ ID NO: 18 QVQLVESGGGSVQPGGSLRLSCTASGWTFRNSAYNLGWFRQAPGQER EAVAAISWRGGSTYYADSVKGRFTISRDNAKNTVTLQMNNLKPEDTA IYYCAADTSARAALYSTGYEYDHWGQGTQVTVSS SEQ ID NO: 19 QVQLVETGGGLVQAGGSLRLSCAASGRTFNNDVMAWFRQAPGTERE FVALITAGGGTHYADSVKGRFVISRDADKNMAYLQMNSLKSEDTAIY YCGADENPPGWPSRWSSAYDYWGQGTQVTVSS SEQ ID NO: 20 QVQLVETGGGLVQAGGSLRLSCAASGRTFNNDVMAWFRQAPGTERE FVALITAGGGTHYADSVKGRFVISRDNDKNMAYLQMNSLKSEDTAIY YCGADEAPPGWPSRWSSAYDYWGQGTQVTVSS SEQ ID NO: 21 QVQLVETGGGLVQAGGSLRLSCAASGRTFNNDVMAWFRQAPGTERE FVALITAGGGTHYADSVKGRFVISRDNDKNMAYLQMNSLKSEDTAIY YCGADENAPGWPSRWSSAYDYWGQGTQVTVSS SEQ ID NO: 22 QVQLVETGGGLVQAGGSLRLSCAASGRTFNNDVMAWFRQAPGTERE FVALITAGGGTAYADSVKGRFVISRDNDKNMAYLQMNSLKSEDTAIY YCGADENPPGWPSRWSSAYDYWGQGTQVTVSS SEQ ID NO: 23 QVQLVETGGGLVQAGGSLRLSCAASGRTFNNDVMAWFRQAPGTERE FVALITAGGGTHYADSVKGRFVISRDNDKNMAYLQMNSLKSEDTAIY YCGADENPAGWPSRWSSAYDYWGQGTQVTVSS SEQ ID NO: 24 QVQLVETGGGLVQAGGSLRLSCAASGRTFNADVMAWFRQAPGTERE FVALITAGGGTHYADSVKGRFVISRDNDKNMAYLQMNSLKSEDTAIY YCGADENPPGWPSRWSSAYDYWGQGTQVTVSS SEQ ID NO: 25 QVQLVETGGGLVQAGGSLRLSCAASGRTFNNDVMAWFRQAPGTERE FVALITAGGGTHYADSVKGRFVISRDNDKNMAYLQMNSLKSEDTAIY YCGADENPPGWASRWSSAYDYWGQGTQVTVSS SEQ ID NO: 26 QVQLVETGGGLVQAGGSLRLSCAASGRTFNNDVMAWFRQAPGTERE FVALITAGGGTHYADSVKGRFVISRDNDKNMAYLQMNSLKSEDTAIY YCGADENPPGAPSRWSSAYDYWGQGTQVTVSS SEQ ID NO: 27 QVQLVETGGGLVQAGGSLRLSCAASGRTFNNDVMAWFRQAPGTERE FVALIGAGGGTHYADSVKGRFVISRDNDKNMAYLQMNSLKSEDTAIY YCGADENPPGWPSRWSSAYDYWGQGTQVTVSS SEQ ID NO: 28 QVQLVESGGGSVQPGGSLRLSCTASGWTFRDSAYNLGWFRQAPGQER EAVAAISWRGGSTYYADSVKGRFTISRDNAKNTVTLQMNNLKPEDTA IYYCAADTSARAALASTGYEYDHWGQGTQVTVSSSEQ ID NO: 29 QVQLVESGGGSVQPGGSLRLSCTASGWTFRDSAYALGWFRQAPGQER EAVAAISWRGGSTYYADSVKGRFTISRDNAKNTVTLQMNNLKPEDTA IYYCAADTSARAALYSTGYEYDHWGQGTQVTVSS SEQ ID NO: 30 QVQLVESGGGSVQPGGSLRLSCTASGWAFRDSAYNLGWFRQAPGQE REAVAAISWRGGSTYYADSVKGRFTISRDNAKNTVTLQMNNLKPEDT AIYYCAADTSARAALYSTGYEYDHWGQGTQVTVSS SEQ ID NO: 31 QVQLVESGGGSVQPGGSLRLSCTASGWTFADSAYNLGWFRQAPGQER EAVAAISWRGGSTYYADSVKGRFTISRDNAKNTVTLQMNNLKPEDTA IYYCAADTSARAALYSTGYEYDHWGQGTQVTVSS SEQ ID NO: 32 QVQLVESGGGSVQPGGSLRLSCTASGWTFRDSAYNLGWFRQAPGQER EAVAAISWRGGSTYYADSVKGRFTISRDNAKNTVTLQMNNLKPEDTA IYYCAADTSARAALYSTAYEYDHWGQGTQVTVSS SEQ ID NO: 33 QVQLVESGGGSVQPGGSLRLSCTASGATFRDSAYNLGWFRQAPGQER EAVAAISWRGGSTYYADSVKGRFTISRDNAK NTVTLQMNNLKPEDTAIYYCAADTSARAALYSTGYEYDHWGQGTQV TVSS SEQ ID NO: 34 QVQLVESGGGSVQPGGSLRLSCTASGWTFRDSAYNLGWFRQAPGQER EAVAAISWRGGSTYYADSVKGRFTISRDNAKNTVTLQMNNLKPEDTA IYYCAADTSAAAALYSTGYEYDHWGQGTQVTVSS SEQ ID NO: 35 QVQLVESGGGSVQPGGSLRLSCTASGWTARDSAYNLGWFRQAPGQE REAVAAISWRGGSTYYADSVKGRFTISRDNAKNTVTLQMNNLKPEDT AIYYCAADTSARAALYSTGYEYDHWGQGTQVTVSS SEQ ID NO: 36 QVQLVESGGGSVQPGGSLRLSCTASGWTFRDSAYNLGWFRQAPGQER EAVAAISWRGGSTYYADSVKGRFTISRDNAKNTVTLQMNNLKPEDTA IYYCAADTAARAALYSTGYEYDHWGQGTQVTVSS SEQ ID NO: 37 QVQLVESGGGSVQPGGSLRLSCTASGWTFRDSAANLGWFRQAPGQER EAVAAISWRGGSTYYADSVKGRFTISRDNAKNTVTLQMNNLKPEDTA IYYCAADTSARAALYSTGYEYDHWGQGTQVTVSS SEQ ID NO: 38 QVQLVESGGGSVQPGGSLRLSCTASGWTFRDSAYNLGWFRQAPGQER EAVAAISWRGGSTYYADSVKGRFTISRDNAKNTVTLQMNNLKPEDTA IYYCAADASARAALYSTGYEYDHWGQGTQVTVSS SEQ ID NO: 39 QVQLVESGGGSVQPGGSLRLSCTASGWTFRDSAYNLGWFRQAPGQER EAVAAISWRGGSTYYADSVKGRFTISRDNAKNTVTLQMNNLKPEDTA IYYCAADTSARAALYSTGAEYDHWGQGTQVTVSS SEQ ID NO: 40 QVQLVESGGGSVQPGGSLRLSCTASGWTFRDSAYNLGWFRQAPGQER EAVAAISWRGGSTYYADSVKGRFTISRDNAKNTVTLQMNNLKPEDTA IYYCAADTSARAALYSTGYAYDHWGQGTQVTVS SEQ ID NO: 41 QVQLVESGGGSVQPGGSLRLSCTASGWTFRDSAYNLGWFRQAPGQER EAVAAISWRGGSTYYADSVKGRFTISRDNAKNTVTLQMNNLKPEDTA IYYCAADTSARAALYSTGYEADHWGQGTQVTVS SEQ ID NO: 42 QVQLVESGGGSVQPGGSLRLSCTASGWTFRDSAYNLGWFRQAPGQER EAVAAISWRGGSTYYADSVKGRFTISRDNAKNTVTLQMNNLKPEDTA IYYCAADTSARAALYSTGYEYAHWGQGTQVTVSS

[0158] As mentioned herein, other C1q complement factor-engaging domains may for example be suitably chosen from the C1q binders listed in Table A that have a reduced affinity for C1q (as described herein). In particular, the C1q complement factor-engaging domains or polypeptides described herein may be chosen from the variants of Nb75 in Table A that have a reduced affinityfor C1q and the variants of Nb78 in Table A that have a reduced affinity for C1q. For example, as further described herein, they may be chosen from the variants of Nb75 in Table A that have one or more mutations in their CDRs (which mutations are such that they reduce the affinity for C1q, compared to the affinity for C1q of Nb75, as further described herein) or from the variants of Nb78 in Table A that have one or more mutations in their CDRs (which mutations are such that they reduce the affinity for C1q, compared to the affinity for C1q of Nb78, as further described herein). According to one specific but non-limiting aspect, which is as further herein, the C1q complement factor-engaging domain(s) may be chosen from the variants of Nb78 in Table A that have one or more alanine mutations in their CDRs (which alanine mutations are such that they reduce the affinity for C1q, compared to the affinity for C1q of Nb78, as further described herein).

[0159] In some embodiments, the complement factor-engaging domain comprises one or more modifications compared to the wild-type C1q complement factor-engaging polypeptide of SEQ ID NO: 3. In some embodiments, the complement factor-engaging domain comprises one or more amino acid substitutions compared to the wild-type C1q complement factor-engaging polypeptide of SEQ ID NO: 3. In some embodiments, the complement factor-engaging domain binds C1q, but binds C1q with reduced affinity as compared to the binding affinity of the polypeptide of SEQ ID NO: 3 for C1q. In some embodiments, the complement factor-engaging domain that binds C1q comprises a N73A amino acid substitution, relative to a wild-type C1q-engaging polypeptide having an amino acid sequence of SEQ ID NO: 3. In other embodiments, the complement factor- engaging domain has a N100A amino acid substitution relative to a wild-type C1q-engaging polypeptide having an amino acid sequence of SEQ ID NO: 3. In some embodiments, the complement factor-engaging domain has a P101A amino acid substitution relative to a wild-type C1q-engaging polypeptide having an amino acid sequence of SEQ ID NO: 3. In other embodiments, the complement factor-engaging domain has a H58A amino acid substitution, relative to a wild-type C1q-engaging polypeptide having an amino acid sequence of SEQ ID NO: 3. In some embodiments, the complement factor-engaging domain has a P102A amino acid substitution, relative to a wild-type C1q-engaging polypeptide having an amino acid sequence of SEQ ID NO: 3. In some embodiments, the complement factor-engaging domain has a N31A amino acid substitution, relative to a wild-type C1q-engaging polypeptide having an amino acid sequence of SEQ ID NO: 3. In some embodiments, the complement factor-engaging domain has a P105A amino acid substitution, relative to a wild-type C1q-engaging polypeptide having an amino acid sequence of SEQ ID NO: 3. In some embodiments, the complement factor-engaging domain has a W104A amino acid substitution, relative to a wild-type C1q-engaging polypeptide having an amino acid sequence of SEQ ID NO: 3. In some embodiments, the complement factor-engaging domain has a T52G amino acid substitution, relative to a wild-type C1q-engaging polypeptide having an amino acid sequence of SEQ ID NO: 3. In some embodiments, the complement factor-engaging domain that binds C1q comprises any one or more of the above amino acid substitutions relative to SEQ ID NO: 3. In some embodiments, the complement factor- engaging domain that binds C1q comprises one or more amino acid substitutions relative to SEQ ID NO: 3, wherein the one or more amino acid substitutions are selected from the group consisting of N73A, N100A, P101A, H58A, P102A, N31A, P105A, W104A, and T52G. In some embodiments, the complement factor-engaging domain that binds C1q comprises one or more amino acid substitutions relative to SEQ ID NO: 3, wherein the one or more amino acid substitutions comprise any one or more of N73A, N100A, P101A, H58A, P102A, N31A, P105A, W104A, and T52G. In some embodiments, the complement factor-engaging domain that binds C1q comprises a total of no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9 or no more than 10 amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 3, e.g., where the one or more amino acid substitutions are selected from, or comprise, any one or more of N73A, N100A, P101A, H58A, P102A, N31A, P105A, W104A, and T52G. In some embodiments, the complement factor-engaging domain that binds C1q comprises any one or more of the above amino acid substitutions relative to SEQ ID NO: 3, and has a total of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the complement factor-engaging domain that binds C1q comprises any one or more of the above amino acid substitutions relative to SEQ ID NO: 3, and has a sequence that has at least 70%, 80%, 90%, 95%, 97%, 99% sequence identity to SEQ ID NO: 3. In any of these embodiments, the complement factor-engaging domain binds C1q, but binds C1q with reduced affinity as compared to the binding affinity of the polypeptide of SEQ ID NO: 3 for C1q. In some embodiments, the C1q is human C1q. In some embodiments, the binding affinity is assessed by a method as described herein, such as by biolayer interferometry.

[0160] In some embodiments, the complement factor-engaging domain comprises one or more modifications compared to the wild-type C1q complement factor-engaging polypeptide of SEQ ID NO: 2. In some embodiments, the complement factor-engaging domain comprises one or more amino acid substitutions compared to the wild-type C1q complement factor-engaging polypeptide of SEQ ID NO: 2. In some embodiments, the complement factor-engaging domain binds C1q, but binds C1q with reduced affinity as compared to the binding affinity of the polypeptide of SEQ ID NO: 2 for C1q. In some embodiments, the one or more amino acid substitutions compared to the wild-type C1q complement factor-engaging polypeptide of SEQ ID NO: 2 are at positions selectedfrom residue 32 to residue 114 of SEQ ID NO: 2. In some embodiments, the complement factor- engaging domain comprises one or more amino acid substitutions at positions selected from residue 32 to residue 114 relative to SEQ ID NO: 2. In some embodiments, the one or more amino acid substitutions compared to the wild-type C1q complement factor-engaging polypeptide of SEQ ID NO: 2 are at positions selected from residue 32, residue 53, residue 59, residue 74, residue 101, residue 102, residue 103, residue 104, residue 105, residue 106, residue 113, and residue 114 of SEQ ID NO: 2. In some embodiments, the complement factor-engaging domain comprises one or more amino acid substitutions at positions selected from residue 32, residue 53, residue 59, residue 74, residue 101, residue 102, residue 103, residue 104, residue 105, residue 106, residue 113, and residue 114, relative to SEQ ID NO: 2. In some embodiments, the one or more amino acid substitutions compared to the wild-type C1q complement factor-engaging polypeptide of SEQ ID NO: 2 are at positions selected from residue 101, residue 102, residue 113, and residue 114 of SEQ ID NO: 2. In some embodiments, the complement factor-engaging domain comprises one or more amino acid substitutions at positions selected from residue 101, residue 102, residue 113, and residue 114, relative to SEQ ID NO: 2. In some embodiments, the complement factor-engaging domain has a Y113A amino acid substitution relative to a C1q-engaging polypeptide having an amino acid sequence of SEQ ID NO: 2. In some embodiments, the complement factor-engaging domain has an E114A amino acid substitution relative to a C1q-engaging polypeptide having an amino acid sequence of SEQ ID NO: 2. In some embodiments, the complement factor-engaging domain has a D101A amino acid substitution relative to a C1q-engaging polypeptide having an amino acid sequence of SEQ ID NO: 2. In some embodiments, the complement factor-engaging domain has a T102A amino acid substitution and a Y113A amino acid substitution, relative to a C1q-engaging polypeptide having an amino acid sequence of SEQ ID NO: 2. In some embodiments, the complement factor-engaging domain has a T102A amino acid substitution and an E114A amino acid substitution, relative to a C1q-engaging polypeptide having an amino acid sequence of SEQ ID NO: 2. In some embodiments, the complement factor-engaging domain comprises any one or more of the above amino acid substitutions relative to SEQ ID NO: 2. In some embodiments, the complement factor-engaging domain comprises one or more amino acid substitutions relative to SEQ ID NO: 2, wherein the one or more amino acid substitutions are selected from the group consisting of Y113A, E114A, D101A, and T102A. In some embodiments, the complement factor-engaging domain comprises one or more amino acid substitutions relative to SEQ ID NO: 2, wherein the one or more amino acid substitutions comprise any one or more of Y113A, E114A, D101A, and T102A. In some embodiments, the complement factor-engaging domain comprises the Y113A amino acid substitution, relative to the amino acid sequence of SEQID NO: 2, and comprises an amino acid sequence with about 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 4. In some embodiments, the complement factor-engaging domain comprises the E114A amino acid substitution, relative to the amino acid sequence of SEQ ID NO: 2, and comprises an amino acid sequence with about 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 5. In some embodiments, the complement factor-engaging domain comprises the D101A amino acid substitution, relative to the amino acid sequence of SEQ ID NO: 2, and comprises an amino acid sequence with about 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 6. In some embodiments, the complement factor-engaging domain comprises the amino acid substitutions T102A and Y113A, relative to the amino acid sequence of SEQ ID NO: 2, and comprises an amino acid sequence with about 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 7. In some embodiments, the complement factor- engaging domain comprises the amino acid substitutions T102A and E114A, relative to the amino acid sequence of SEQ ID NO: 2, and comprises an amino acid sequence with about 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 8. In some embodiments, the complement factor-engaging domain comprises a total of no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9 or no more than 10 amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 2, e.g., where the amino acid substitutions are selected from, or comprise, amino acid substitutions at positions corresponding to a position in SEQ ID NO: 2 ranging from residue 32 to residue 114; wherein the amino acid substitutions are selected from, or comprise, amino acid substitutions at positions corresponding to residue 32, residue 53, residue 59, residue 74, residue 101, residue 102, residue 103, residue 104, residue 105, residue 106, residue 113, residue 114 of SEQ ID NO: 2; wherein the amino acid substitutions are selected from, or comprise, amino acid substitutions at positions corresponding to residue 101, residue 102, residue 113 and residue 114 of SEQ ID NO: 2; or wherein the amino acid substitutions are selected from, or comprise, any one or more of Y113A, E114A, D101A, and T102A, relative to SEQ ID NO: 2. In some embodiments, the complement factor-engaging domain comprises any one or more of the above amino acid substitutions relative to SEQ ID NO: 2, and has a total of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the complement factor-engaging domain comprises any one or more of the above amino acid substitutions relative to SEQ ID NO: 2, and has a sequence that has at least 70%, 80%, 90%, 95%, 97%, 99% sequence identity to SEQ ID NO: 2. In any of these embodiments, the complement factor-engaging domain binds C1q, such as human C1q. In any of these embodiments, the complement factor-engaging domain binds C1q, but binds C1q with reduced affinity as comparedto the binding affinity of the polypeptide of SEQ ID NO: 2 for C1q. In some embodiments, the C1q is human C1q. In some embodiments, the binding affinity is assessed by a method as described herein, such as by biolayer interferometry. Bispecific constructs comprising C1q complement factor-engaging domain

[0161] Also disclosed herein are bispecific constructs that comprise a complement factor- engaging domain that binds to C1q complement factor. In particular, disclosed are bispecific constructs that comprise a complement factor-engaging domain that binds to C1q complement factor with optimized affinity. In some embodiments, the complement factor-engaging domain that binds to C1q is a complement factor-engaging domain as described herein, such as a C1q complement factor-engaging domain as described herein. In some embodiments, the complement factor-engaging domain is an optimized or engineered complement factor-engaging domain as described herein (e.g., a complement factor-engaging domain having an amino acid sequence of any one of SEQ ID NO: 4 – SEQ ID NO: 42). In some embodiments, the complement factor- engaging domain is chosen among (engineered or non-engineered) C1q-binding domains having the optimized C1q binding affinity that results in improved CDC in the bispecific construct. In some embodiments, the C1q complement factor-engaging domain can bind C1q with high affinity. In some embodiments, the C1q complement factor-engaging domain can bind C1q with low affinity. In some embodiments, the C1q complement factor-engaging domain comprises one or more modifications relative to a corresponding wild-type C1q complement factor-engaging polypeptide, said one or more modifications resulting in reduced binding affinity to a C1q complement factor as compared to a binding affinity of the corresponding wild-type C1q complement factor-engaging polypeptide to the C1q complement factor. In some embodiments, the C1q complement factor-engaging domain cannot bind C1q directly. A non-limiting example of a complement factor C1q recognized by bispecific constructs of the present disclosure is human C1q, including the three polypeptide chains A, B, and C:

[0162] C1q, chain A (Homo sapiens), Accession No. Protein Data Base: NP_057075.1; GenBank No.: NM_015991: gi|7705753|ref|NP_057075.1| complement C1q subcomponent subunit A precursor [Homo sapiens]. MEGPRGWLVLCVLAISLASMVTEDLCRAPDGKKGEAGRPGRRGRPGLKGEQ GEPGAPGIRTGIQGLKGDQGEPGPSGNPGKVGYPGPSGPLGARGIPGIKGT KGSPGNIKDQPRPAFSAIRRNPPMGGNVVIFDTVITNQEEPYQNHSGRFVC TVPGYYYFTFQVLSQWEICLSIVSSSRGQVRRSLGFCDTTNKGLFQVVSGG MVLQLQQGDQVWVEKDPKKGHIYQGSEADSVFSGFLIFPSA (SEQ ID NO: 43)

[0163] C1q, chain B (Homo sapiens), Accession No. Protein Data Base: NP_000482.3; GenBank No.: NM_000491.3: >gi|87298828|ref|NP_000482.3| complement C1q subcomponent subunit B precursor [Homo sapiens] . MMMKIPWGSIPVLMLLLLLGLIDISQAQLSCTGPPAIPGIPGIPGTPGPDG QPGTPGIKGEKGLPGLAGDHGEFGEKGDPGIPGNPGKVGPKGPMGPKGGPG APGAPGPKGESGDYKATQKIAFSATRTINVPLRRDQTIREDHVITNMNNNY EPRSGKFTCKVPGLYYFTYHASSRGNLCVNLMRGRERAQKVVTFCDYAYNT FQVTTGGMVLKLEQGENVFLQATDKNSLLGMEGANSIFSGFLLFPDMEA (SEQ ID NO: 44)

[0164] C1q, chain C (Homo sapiens), Accession No. Protein Data Base: NP_001107573.1; GenBank No.: NM_001114101.1: >gi|166235903|ref|NP_001107573.1| complement C1q subcomponent subunit C precursor [Homo sapiens]. MDVGPSSLPHLGLKLLLLLLLLPLRGQANTGCYGIPGMPGLPGAPGKDGYDG LPGPKGEPGIPAIPGIRGPKGQKGEPGLPGHPGKNGPMGPPGMPGVPGPMGI PGEPGEEGRYKQKFQSVFTVTRQTHQPPAPNSLIRFNAVLTNPQGDYDTSTG KFTCKVPGLYYFVYHASHTANLCVLLYRSGVKVVTFCGHTSKTNQVNSGGVL LRLQVGEEVWLAVNDYYDMVGIQGSDSVFSGFLLFPD (SEQ ID NO: 45)

[0165] Accordingly, a C1q complement factor-engaging domain of the present disclosure (or corresponding bispecific construct or C1q complement factor-engaging polypeptide) may bind to polypeptide chain A, polypeptide chain B, and / or polypeptide chain C of a C1q complement factor. In some embodiments, a C1q complement factor-engaging domain according to the present disclosure (or corresponding bispecific construct or C1q complement factor-engaging polypeptide) binds to polypeptide chain A, polypeptide chain B, and / or polypeptide chain C of human C1q or a homolog thereof, such as mouse, rat, rabbit, monkey, dog, cat, cow, horse, camel, sheep, goat, or pig C1q.

[0166] In some embodiments, the C1q complement factor-engaging polypeptide or C1q complement factor-engaging domain comprises a variable domain of heavy chain antibody (VHH) domain (sometimes referred to as a camelid antibody). In some embodiments, the VHH domain of the C1q complement factor-engaging domain binds to the C1q complement factor, wherein the C1q complement factor is a human C1q complement factor or a cynomolgus monkey C1q complement factor. In some embodiments, the VHH domain of the C1q complement factor- engaging polypeptide or domain binds to the C1q complement factor with a KD of about 1 nM to about 10 μM. In some embodiments, the VHH domain of the C1q complement factor-engaging polypeptide or domain binds to the C1q complement factor with a KD of about 1 nM to about 5 μM. In some embodiments, the VHH domain of the C1q complement factor-engaging polypeptide or domain binds to the C1q complement factor with a KD of about 10 nM to about 2 μM. In some embodiments, the VHH domain of the C1q complement factor-engaging polypeptide or domainbinds to the C1q complement factor with a KD of about 100 nM to about 1.5 μM. In some embodiments, the VHH domain of the C1q complement factor-engaging polypeptide or domain binds to the C1q complement factor with a KD of about 100 nM, about 150 nM, about 200 nM, about 250 nM, about 300 nM, about 350 nM, about 400 nM, , about 450 nM, about 500 nM, about 550 nM, about 600 nM, about 650 nM, about 700 nM, , about 750 nM, about 800 nM, about 850 nM, about 900 nM, about 950 nM, about 1000 nM, about 1100 nM, about 1200 nM, about 1300 nM, about 1400 nM, or about 1500 nM. In some cases, the binding affinity is measured by biolayer interferometry.

[0167] In some embodiments, the C1q complement factor-engaging polypeptide or C1q complement factor-engaging domain binds to a C1q complement factor with a binding affinity of from about 100 nM to about 10 μM, from about 200 nM to about 10 μM, from about 300 nM to about 10 μM, from about 400 nM to about 10 μM, from about 500 nM to about 10 μM, from about 600 nM to about 10 μM, from about 700 nM to about 10 μM, from about 800 nM to about 10 μM, from about 900 nM to about 1 μM, from about 1 μM to about 10 μM, from about 1.1 μM to about 10 μM, from about 1.2 μM to about 10 μM, from about 1.3 μM to about 10 μM, from about 1.4 μM to about 10 μM, from about 1.5 μM to about 10 μM, from about 1.6 μM to about 10 μM, from about 1.7 μM to about 10 μM, from about 1.8 μM to about 10 μM, from about 1.9 μM to about 10 μM, or from about 2 μM to about 10 μM. In some embodiments, the C1q complement factor- engaging polypeptide or complement factor-engaging domain binds to a C1q complement factor with a binding affinity of about 1 μM. In some cases, the binding affinity is measured by biolayer interferometry. In some embodiments, the C1q complement factor-engaging polypeptide or domain binds to a C1q complement factor, such as a human C1q complement factor, with a binding affinity of 0.9 to 1.1 μM, or 0.95 to 1.05 μM, or 0.99 to 1.01 μM, as determined by biolayer interferometry.

[0168] In some embodiments, the C1q complement factor-engaging polypeptide or complement factor-engaging domain is a single domain antibody. In some embodiments, the C1q complement factor-engaging polypeptide or complement factor-engaging domain comprises a single domain antibody. In some embodiments, the single domain antibodies provided herein comprise an antigen binding site in a single polypeptide that binds to a C1q complement factor. In some embodiments, the single domain antibody is a nanobody. The single chain antibodies disclosed herein may, in certain embodiments be bispecific or multispecific single domain antibodies as described elsewhere herein, where two single domain antibodies are coupled. Single domain antibodies can also be smaller than Fab fragments (~50 kDa) of an antibody that comprises one light chain and half a heavy chain. The single domain antibodies (such as VHH domains) used in the presentinvention preferably comprise one or more CDRs, such as 3 CDRs. In particular, the CDRs may identify the specificity of the antibody and accordingly it is preferred that the C1q complement factor-engaging polypeptide or complement factor-engaging domain comprises one or more CDRs, preferably at least 1, more preferably at least 2, yet more preferably 3 or more CDRs. In some embodiments, the C1q complement factor-engaging polypeptide or complement factor- engaging domain comprises 3 CDRs.

[0169] The bispecific constructs as described herein can comprise a single domain antibody that can be used to recruit and activate the complement cascade to specific targets, and thereby specifically activate an innate immune response at a specific target. Such bispecific constructs are therefore particularly useful in medicine for use as a medicament, and particularly for use in treatment of a disease. In some embodiments, the bispecific constructs are capable of specifically binding to an epitope of human complement factor C1q and / or the proteolytic derivatives. In some embodiments, the bispecific constructs as described herein can directly activate the complement system. In some embodiments, the bispecific constructs as described herein can activate complement by binding to C1q complement factor. In some embodiments, the bispecific constructs as disclosed herein comprise a C1q engaging moiety that can bind directly to C1q. In some embodiments, the C1q engaging moiety that can bind directly to C1q can promote a targeted complement-mediated immunological response. In other embodiments, the bispecific constructs as described herein can indirectly activate the complement system. In some embodiments, the bispecific constructs as described herein can activate complement without binding to C1q complement factor. In some embodiments, the bispecific constructs as disclosed herein comprise a C1q engaging moiety that does not bind directly to C1q. In some embodiments, the C1q engaging moiety that does not bind directly to C1q can promote a targeted complement-mediated immunological response. Bispecific constructs comprising an antigen-binding moiety

[0170] The bispecific constructs as described herein comprise an antigen-binding moiety. In some embodiments, the antigen-binding moiety comprises a single domain antibody, a heavy-chain only antibody (HCAb), a single chain antigen-binding fragment (ScFab), a fragment antigen-binding(e.g., Fab, Fab , Fab -SH, F(ab )2) domain, a fragment crystallizable (Fc) domain, a single chainvariable fragment (e.g. scFv), single-chain antibody molecules, a minibody, an antibody, diabodies, or linear antibodies. In some embodiments, the antigen-binding moiety comprises an antibody, an antibody fragment, or antibody mimetic protein. In some embodiments, the antibody is a monoclonal antibody, a bispecific antibody, a polyclonal antibody, or an antibody fragment.In some embodiments, the antibody is an immunoglobulin. In some embodiments, the immunoglobulin comprises a heavy chain, including but not limited to: (IgA), (IgD), (IgE), (IgG), or (IgM), some of which may be further divided into subtypes, e.g., 1 (IgG1), 2 (IgG2), 3 (IgG3), 4 (IgG4), 1 (IgA1) and 2 (IgA2). The light chain of an immunoglobulin may beassigned to one of two types, called kappa ( ) and lambda ( ), based on the amino acid sequenceof its constant domain. In some embodiments, the immunoglobulin comprises two Fab molecules and an Fc domain, linked via an immunoglobulin hinge region. In some embodiments, the antigen- binding moiety comprises Fab. In some embodiments, the antigen-binding moiety is linked to Fab on the N-terminus of a light chain on the Fab. In some embodiments, the antigen-binding moiety is linked to Fab on the N-terminus of a heavy chain on the Fab. In some embodiments, the antigen- binding moiety is linked to Fab on the C-terminus of a light chain on the Fab. In some embodiments, the antigen-binding moiety is linked to Fab on the C-terminus of a heavy chain on the Fab.

[0171] In some embodiments, the bispecific constructs comprise an antigen-binding moiety that binds to a target antigen, such as a target protein. In some embodiments, the target antigen is a protein on a cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the mammalian cell is a cancer cell, an immune cell, an endothelial cell, an epithelial cell, or a microbial cell. In some embodiments, the cancer cell comprises a breast cancer cell, a carcinoma cell, an ovarian cancer cell, an endometrial cancer cell, a colon cancer cell, a gastric cancer cell, a neurological cancer cell, a skin cancer, a lung cancer, or a bladder cancer. In some embodiments, the cancer cell is a carcinoma cell, a breast cancer cell, an ovarian cancer cell, a gastric cancer cell, or a colon cancer cell. In some embodiments, the cancer cell is a lymphoma cell, a myeloma cell, or a leukemia cell. In some embodiments, the target antigen is a protein implicated in a disease. In some embodiments, the disease is an autoimmune disease.

[0172] In some embodiments, the antigen-binding moiety binds to a cell-specific marker, where the cell-specific marker is a cancer-specific marker. In some cases, the cancer-specific marker is expressed on a cancer cell. In some embodiments, the antigen-binding moiety binds to the cancer- specific marker expressed on the cancer cell with a binding affinity that is greater than the binding affinity of the complement factor-engaging domain to C1q. For example, the antigen-binding moiety can bind to the cancer specific marker with a binding affinity of less than 0.1 μM, while the complement factor-engaging domain can bind to C1q with a binding affinity that is greater than or about 0.1 μM, as determined by biolayer interferometry. some embodiments, the cancer- specific marker comprises a protein that is dysregulated in several cancer forms. In some embodiments, the cancer-specific marker comprises a protein that is upregulated in several cancerforms. In other embodiments, the cancer-specific marker comprises a protein that is downregulated in several cancer forms. In some embodiments, the cancer specific marker is CD19, CD20, CD22, CD30, CD33, CD38, CD52, cMET, EGFR, EpCAM, Erb2, FOLR1, GD3, HER, PSMA, PSMA, or VEGF. In some embodiments, the cancer specific marker is EGFR, CD38, CD19, CD20, CD55, CD59, CD7, HER2, EGFR, EpCAM, or FOLR1. In some embodiments, the cancer specific marker is CD7, CD19, CD20, CD38, CD55, CD59, EGFR, EpCAM, FOLR1, or HER2. In some embodiments, the cancer specific marker is CD7, CD19, CD20, CD38, EGFR, EpCAM, FOLR1 or HER2. In some embodiments, the cancer-specific marker is CD19, CD20, CD38, or EGFR. In some embodiments, the cancer specific marker is a tumor marker. As used herein, a tumor marker is anything present in or produced by cancer cells or other cells of the body in response to cancer or certain benign (noncancerous) conditions that provides information about a cancer, such as how aggressive it is, what kind of treatment it may respond to, or whether it is responding to treatment.

[0173] In some embodiments, the bispecific constructs provided herein, when administered to a subject or a population of cells, results in an increase in complement-dependent cytotoxicity (CDC) relative to a reference bispecific construct (for example, a bispecific construct that is otherwise identical but does not comprise one or more modifications to the amino acid sequence of the complement factor-engaging domain, as described herein) or a non-bispecific construct. In some embodiments, the bispecific constructs provided herein, when administered to a population of cells, increase the lysis of cells relative to a reference or non-bispecific construct. In some embodiments, the bispecific constructs provided herein, when administered to a subject, increase the lysis of tumor cells relative to a reference or non-bispecific construct. Linkers

[0174] The bispecific constructs as disclosed herein comprise a complement factor-engaging domain and an antigen-binding moiety. In some cases, the complement factor-engaging domain and antigen binding moiety are linked via a polypeptide linker sequence. In some embodiments, the complement factor-engaging domain is linked or fused to the N-terminal of the antigen binding moiety. In other embodiments, the complement factor-engaging domain is linked or fused to the C-terminal of the antigen binding moiety.

[0175] In some embodiments, the antigen-binding moiety is directly linked to the complement factor-engaging domain. In other embodiments, the antigen-binding moiety is indirectly linked to the complement factor-engaging domain. In some embodiments, the complement factor-engaging domain is linked to an antigen binding moiety through a linker sequence, a Fab region, Fc region, or an IgG region of an antibody.

[0176] In some embodiments, the antigen-binding moiety is linked to the or each complement factor-engaging domain via a linker sequence. In some embodiments, the linker sequence is at least 5 amino acids, at least 10 amino acids, at least 15 amino acids, at least 20 amino acids, at least 25 amino acids, at least 30 amino acids, at least 35 amino acids, at least 40 amino acids, at least 45 amino acids, or at least 50 amino acids. In some embodiments, the linker sequence is about 5 amino acids, about 10 amino acids, about 15 amino acids, about 20 amino acids, about 25 amino acids, about 30 amino acids, about 35 amino acids, about 40 amino acids, about 45 amino acids, or about 50 amino acids. In some embodiments, the linker sequence is about 15 amino acids. In some embodiments, the linker is 15 amino acids. In some embodiments, the linker sequence is about 20 amino acids. In some embodiments, the linker sequence is 20 amino acids.

[0177] In some embodiments, the linker sequence is a non-immunogenic linker peptide. For example, suitable, non-immunogenic linker peptides are, (G4S)n, (SG4)n or G4(SG4)n peptide linkers, wherein "n" is generally a number between 1 and 10, typically between 2 and 4, in particular 2, e.g., the peptides selected from the group consisting of: GGGGS (SEQ ID NO: 46), GGGGSGGGGS (SEQ ID NO: 47), SGGGGSGGGG (SEQ ID NO: 48) and GGGGSGGGGSGGGG (SEQ ID NO: 49), GSPGSSSSGS (SEQ ID NO: 50), (G4S) (SEQ ID NO: 51), (G4S)2(SEQ ID NO: 52), (G4S)3(SEQ ID NO: 53), (G4S)4(SEQ ID NO: 54), GSGSGSGS (SEQ ID NO: 55), GSGSGNGS (SEQ ID NO: 56), GGSGSGSG (SEQ ID NO: 57), GGSGSG (SEQ ID NO: 58), GGSG (SEQ ID NO: 59), GGSGNGSG (SEQ ID NO: 60), GGNGSGSG (SEQ ID NO: 61), and GGNGSG (SEQ ID NO: 62).

[0178] In some embodiments, the bispecific constructs as disclosed herein can enhance the anti- cancer effects of an antibody or fragment thereof. In other embodiments, the bispecific constructs can enhance the anti-inflammatory of an antibody or fragment thereof. In some embodiments, the antibody or fragment thereof is selected from the group consisting of: abagovomab, abciximab, abituzumab, abrilumab, actinium Ac-225 lintuzumab, actoxumab, adalimumab, adecatumumab, aducanumab, afelimomab, afutuzumab, alacizumab pegol, alemtuzumab, alirocumab, altumomab pentetate, amatuximab, anatumomab mafenatox, anetumab ravtansine, anifrolumab, anrukinzumab, apolizumab, sevacizumab, arcitumomab, ascrinvacumab, aselizumab, atezolizumab, atinumab, atorolimumab, avelumab, bapineuzumab, basiliximab, bavituximab, bectumomab, begelomab, belimumab, benralizumab, bertilimumab, besilesomab, 177Lu- tetraxetan-tetulomab, bevacizumab, bezlotoxumab, biciromab, bimagrumab, bimekizumab, bivatuzumab mertansine, blinatumomab, blosozumab, bococizumab, brentuximab vedotin, BrevaRex, briakinumab, brodalumab, brolucizumab, brontictuzumab, canakinumab, cantuzumab mertansine, cantuzumab ravtansine, caplacizumab, capromab pendetide, carlumab, catumaxomab,CBR96-doxorubicin immunoconjugate, cedelizumab, certolizumab pegol, cetuximab, citatuzumab bogatox, cixutumumab, clazakizumab, clenoliximab, clivatuzumab tetraxetan, Clone 003 and Clone 024 (see WO2006 / 099875), CM313, codrituzumab, coltuximab ravtansine, conatumumab, concizumab, cR6261, crenezumab, dacetuzumab, daclizumab, dalotuzumab, dapirolizumab pegol, daratumumab, darleukin, dectrekumab, demcizumab, denintuzumab mafodotin, denosumab, depatuxizumab, depatuxizumab mafodotin, derlotuximab biotin, detumomab, dinutuximab, diridavumab, dorlimomab aritox, drozitumab, duligotumab, dupilumab, durvalumab, dusigitumab, ecromeximab, eculizumab, edobacomab, edrecolomab, efalizumab, efungumab, eldelumab, elgemtumab, elotuzumab, elsilimomab, emactuzumab, emibetuzumab, enavatuzumab, enfortumab vedotin, enlimomab pegol, enoblituzumab, enokizumab, enoticumab, ensituximab, epitumomab cituxetan, epratuzumab, erlizumab, ertumaxomab, etaracizumab, etrolizumab, evinacumab, evolocumab, exbivirumab, fanolesomab, faralimomab, farletuzumab, fasinumab, felvizumab, felzartamab, fezakinumab, FGFR2 Antibody- Drug Conjugate, Fibromun, ficlatuzumab, figitumumab, firivumab, flanvotumab, fletikumab, fontolizumab, foralumab, foravirumab, fresolimumab, fulranumab, futuximab, galiximab, ganitumab, gantenerumab, gavilimomab, gemtuzumab, gevokizumab, girentuximab, glembatumumab vedotin, golimumab, gomiliximab, guselkumab, ibalizumab, ibritumomab tiuxetan, icrucumab, idarucizumab, igovomab, imalumab, imciromab, imgatuzumab, inclacumab, indatuximab ravtansine, indusatumab vedotin, inebilizumab, infliximab, inolimomab, inotuzumab ozogamicin, intetumumab, ipilimumab, iratumumab, isatuximab, istiratumab, itolizumab, ixekizumab, keliximab, labetuzumab, labetuzumab govitecan, lambrolizumab, lampalizumab, lebrikizumab, lemalesomab, lenzilumab, lerdelimumab, leukotuximab, lexatumumab, libivirumab, lifastuzumab vedotin, ligelizumab, lilotomab satetraxetan, lintuzumab, lirilumab, lodelcizumab, lokivetmab, lorvotuzumab mertansine, lucatumumab, lulizumab pegol, lumiliximab, lumretuzumab, mapatumumab, margetuximab, maslimomab, matuzumab, mavrilimumab, mepolizumab, metelimumab, mezagitamab, milatuzumab, milatuzumab-SN-38, minretumomab, mirvetuximab soravtansine, mitazalimab, mitumomab, mogamulizumab, morolimumab, MORO3080, MORO 3087, MORO3088, motavizumab, moxetumomab, pasudotox, MU1053, muromonab-CD3, nacolomab tafenatox, namilumab, naptumomab estafenatox, narnatumab, natalizumab, nebacumab, necitumumab, nemolizumab, nerelimomab, nesvacumab, nimotuzumab, nivolumab, nofetumomab merpentan, obiltoxaximab, obinutuzumab, ocaratuzumab, ocrelizumab, odulimomab, ofatumumab, olaratumab, olokizumab, omalizumab, onartuzumab, ontuxizumab, opicinumab, oportuzumab monatox, oregovomab, orticumab, otelixizumab, otlertuzumab, oxelumab, ozanezumab, ozoralizumab, pagibaximab, palivizumab,panitumumab, pankomab, PankoMab-GEX, panobacumab, parsatuzumab, pascolizumab, pasotuxizumab, pateclizumab, patritumab, pembrolizumab, pemtumomab, perakizumab, pertuzumab, pexelizumab, pidilizumab, pinatuzumab vedotin, pintumomab, placulumab, polatuzumab vedotin, ponezumab, priliximab, pritoxaximab, pritumumab, quilizumab, racotumomab, radretumab, rafivirumab, ralpancizumab, ramucirumab, ranibizumab, raxibacumab, refanezumab, regavirumab, reslizumab, rilotumumab, rinucumab, risankizumab, rituximab, robatumumab, roledumab, romosozumab, rontalizumab, rovelizumab, ruplizumab, sacituzumab govitecan, samalizumab, sarilumab, satumomab pendetide, secukinumab, seribantumab, setoxaximab, sevirumab, sibrotuzumab, sifalimumab, siltuximab, simtuzumab, siplizumab, sirukumab, sofituzumab vedotin, solanezumab, solitomab, sonepcizumab, sontuzumab, stamulumab, sulesomab, suvizumab, tabalumab, tacatuzumab tetraxetan, tadocizumab, tafasitamab, talizumab, tanezumab, tanibirumab, taplitumomab paptox, tarextumab, tefibazumab, teleukin, telimomab aritox, tenatumomab, teneliximab, teplizumab, teprotumumab, tesidolumab, tetulomab, Thorium-227-Epratuzumab Conjugate, ticilimumab, tigatuzumab, tildrakizumab, tisotumab vedotin, tocilizumab, toralizumab, tosatoxumab, tositumomab, tovetumab, tralokinumab, trastuzumab, trastuzumab deruxtecan, trastuzumab emtansine, tregalizumab, tremelimumab, trevogrumab, tucotuzumab celmoleukin, tuvirumab, ublituximab, ulocuplumab, urelumab, urtoxazumab, utomilumab, ustekinumab, vadastuximab talirine, vandortuzumab vedotin, vantictumab, vanucizumab, vapaliximab, varlilumab, vatelizumab, vedolizumab, veltuzumab, vepalimomab, vesencumab, visilizumab, volociximab, vorsetuzumab mafodotin, votumumab, zalutumumab, zanolimumab, zatuximab, ziralimumab, zolimomab aritox, any fragments thereof, or any biosimilars thereof. In some embodiments, the antibody or fragment thereof comprises daratumumab, MOR202, SAR650984, MU1053, tafasitamab, isatuximab, HexaBody-CD38, or rituximab. In some embodiments, the antibody is daratumumab or a fragment thereof. In some embodiments, the antibody is MOR202 or a fragment thereof. In some embodiments, the antibody is SAR650984 or a fragment thereof. In some embodiments, the antibody or fragment thereof is MU1053. In some embodiments, the antibody is isatuximab or a fragment thereof. In some embodiments, the antibody is HexaBody-CD38 or a fragment thereof. In some embodiments, the antibody is tafasitamab or a fragment thereof. In some embodiments, the antibody is rituximab or a fragment thereof.

[0179] In some embodiments, the anti-TNF-alpha antibody is adalimumab. The heavy chain of adalimumab is provided below in: MEFGLSWLFLVAILKGVQCEVQLVESGGGLVQPGRSLRLSCAASGFTFDDYA MHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSSASTKGPSVFPLAPSSK STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK (SEQ ID NO: 63)

[0180] The light chain of adalimumab is provided below: MDMRVPAQLLGLLLLWLRGARCDIQMTQSPSSLSASVGDRVTITCRASQGIR NYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPED VATYYCQRYNRAPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVC LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY EKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 64)

[0181] In some embodiments, the invention relates to an antibody of the invention (as further described herein) that is directed against TNF alpha. In particular, the invention relates to such an antibody of the invention in which the two heavy chains that are present in said antibody of the invention are both the heavy chain of adalimumab and the two light chains that are present in said antibody of the invention are both the light chain of adalimumab (and which antibody of the invention also contains two C1q binders with reduced affinity for C1q, as further described herein).

[0182] In another embodiment, the invention relates to a polypeptide that comprises the heavy chain of adalimumab, which is linked or fused (as further described herein) to a C1q binder with reduced affinity for C1q (as further described herein); as well as to an antibody or antibody construct that comprises two such heavy chains (and two suitable light chains, that are preferably each the light chain of adalimumab). In another embodiment, the invention relates to a polypeptide that comprises the light chain of adalimumab, which is linked or fused (as further described herein) to a C1q binder with reduced affinity for C1q (as further described herein); as well as to an antibody or antibody construct that comprises two such light chains (and two suitable heavy chains, that are preferably each the heavy chain of adalimumab).

[0183] In some embodiments, the anti-CD52 antibody is alemtuzumab. The heavy chain of alemtuzumab is provided below: QVQLQESGPGLVRPSQTLSLTCTVSGFTFTDFYMNWVRQPPGRGLEWIGFIR DKAKGYTTEYNPSVKGRVTMLVDTSKNQFSLRLSSVTAADTAVYYCAREGHT AAPFDYWGQGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH KPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRT PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 65)

[0184] The light chain of alemtuzumab is provided below: DIQMTQSPSSLSASVGDRVTITCKASQNIDKYLNWYQQKPGKAPKLLIYNTN NLQTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCLQHISRPRTFGQGTKV EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNR (SEQ ID NO: 66)

[0185] In some embodiments, the invention relates to an antibody of the invention (as further described herein) that is directed against CD52. In particular, the invention relates to such an antibody of the invention in which the two heavy chains that are present in said antibody of the invention are both the heavy chain of alemtuzumab and the two light chains that are present in said antibody of the invention are both the light chain of alemtuzumab (and which antibody of the invention also contains two C1q binders with reduced affinity for C1q, as further described herein).

[0186] In another embodiment, the invention relates to a polypeptide that comprises the heavy chain of alemtuzumab, which is linked or fused (as further described herein) to a C1q binder with reduced affinity for C1q (as further described herein); as well as to an antibody or antibody construct that comprises two such heavy chains (and two suitable light chains, that are preferably each the light chain of alemtuzumab). In another embodiment, the invention relates to a polypeptide that comprises the light chain of alemtuzumab, which is linked or fused (as further described herein) to a C1q binder with reduced affinity for C1q (as further described herein); as well as to an antibody or antibody construct that comprises two such light chains (and two suitable heavy chains, that are preferably each the heavy chain of alemtuzumab).

[0187] In some embodiments, the anti-EGFR antibody is cetuximab. The heavy chain of cetuximab is provided below: QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIW SGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDY EFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP SNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 67)

[0188] The light chain of cetuximab is provided below: DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYAS ESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKL ELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC (SEQ ID NO: 68)

[0189] In some embodiments, the invention relates to an antibody of the invention (as further described herein) that is directed against EGFR. In particular, the invention relates to such an antibody of the invention in which the two heavy chains that are present in said antibody of the invention are both the heavy chain of cetuximab and the two light chains that are present in said antibody of the invention are both the light chain of cetuximab (and which antibody of the invention also contains two C1q binders with reduced affinity for C1q, as further described herein).

[0190] In another embodiment, the invention relates to a polypeptide that comprises the heavy chain of cetuximab, which is linked or fused (as further described herein) to a C1q binder with reduced affinity for C1q (as further described herein); as well as to an antibody or antibody construct that comprises two such heavy chains (and two suitable light chains, that are preferably each the light chain of cetuximab). In another embodiment, the invention relates to a polypeptide that comprises the light chain of cetuximab, which is linked or fused (as further described herein) to a C1q binder with reduced affinity for C1q (as further described herein); as well as to an antibody or antibody construct that comprises two such light chains (and two suitable heavy chains, that are preferably each the heavy chain of cetuximab).

[0191] In other embodiments, the anti-EGFR antibody is trastuzumab. The heavy chain of trastuzumab is provided below: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIY PTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGF YAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK PSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 69)

[0192] The light chain of trastuzumab is provided below: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSAS FLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKV EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC (SEQ ID NO: 70)

[0193] In some embodiments, the invention relates to an antibody of the invention (as further described herein) that is directed against EGFR in which the two heavy chains that are present in said antibody of the invention are both the heavy chain of trastuzumab and the two light chains that are present in said antibody of the invention are both the light chain of trastuzumab (and whichantibody of the invention also contains two C1q binders with reduced affinity for C1q, as further described herein).

[0194] In another embodiment, the invention relates to a polypeptide that comprises the heavy chain of trastuzumab, which is linked or fused (as further described herein) to a C1q binder with reduced affinity for C1q (as further described herein); as well as to an antibody or antibody construct that comprises two such heavy chains (and two suitable light chains, that are preferably each the light chain of trastuzumab). In another embodiment, the invention relates to a polypeptide that comprises the light chain of trastuzumab, which is linked or fused (as further described herein) to a C1q binder with reduced affinity for C1q (as further described herein); as well as to an antibody or antibody construct that comprises two such light chains (and two suitable heavy chains, that are preferably each the heavy chain of trastuzumab).

[0195] In some embodiments, the anti-CD38 antibody is daratumumab. The heavy chain of daratumumab is provided below: EVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSAIS GSGGGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCAKDKILWF GEPVFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFP EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 71)

[0196] The light chain of daratumumab is provided below: EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDAS NRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKV EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC (SEQ ID NO: 72)

[0197] In some embodiments, the invention relates to an antibody of the invention (as further described herein) that is directed against CD38. In particular, the invention relates to such an antibody of the invention in which the two heavy chains that are present in said antibody of the invention are both the heavy chain of daratumumab and the two light chains that are present in said antibody of the invention are both the light chain of daratumumab (and which antibody of the invention also contains two C1q binders with reduced affinity for C1q, as further described herein).

[0198] In another embodiment, the invention relates to a polypeptide that comprises the heavy chain of daratumumab, which is linked or fused (as further described herein) to a C1q binder with reduced affinity for C1q (as further described herein); as well as to an antibody or antibodyconstruct that comprises two such heavy chains (and two suitable light chains, that are preferably each the light chain of daratumumab). In another embodiment, the invention relates to a polypeptide that comprises the light chain of daratumumab, which is linked or fused (as further described herein) to a C1q binder with reduced affinity for C1q (as further described herein); as well as to an antibody or antibody construct that comprises two such light chains (and two suitable heavy chains, that are preferably each the heavy chain of daratumumab).

[0199] In other embodiments, the anti-CD38 antibody is isatuximab. The heavy chain of isatuximab is provided below: QVQLVQSGAEVAKPGTSVKLSCKASGYTFTDYWMQWVKQRPGQGLEWIGTIY PGDGDTGYAQKFQGKATLTADKSSKTVYMHLSSLASEDSAVYYCARGDYYGS NSLDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK PSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 73) QVQLVQSGAEVAKPGTSVKLSCKASGYTFTDYWMQWVKQRPGQGLEWIGTIY PGDGDTGYAQKFQGKATLTADKSSKTVYMHLSSLASEDSAVYYCARGDYYGS NSLDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK PSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 74)

[0200] The light chain of isatuximab is provided below: DIVMTQSHLSMSTSLGDPVSITCKASQDVSTVVAWYQQKPGQSPRRLIYSAS YRYIGVPDRFTGSGAGTDFTFTISSVQAEDLAVYYCQQHYSPPYTFGGGTKL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC (SEQ ID NO: 75) DIVMTQSHLSMSTSLGDPVSITCKASQDVSTVVAWYQQKPGQSPRRLIYSAS YRYIGVPDRFTGSGAGTDFTFTISSVQAEDLAVYYCQQHYSPPYTFGGGTKL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC (SEQ ID NO: 76)

[0201] In some embodiments, the invention relates to an antibody of the invention (as further described herein) that is directed against CD38 in which the two heavy chains that are present in said antibody of the invention are both the heavy chain of isatuximab and the two light chains that are present in said antibody of the invention are both the light chain of isatuximab (and whichantibody of the invention also contains two C1q binders with reduced affinity for C1q, as further described herein).

[0202] In another embodiment, the invention relates to a polypeptide that comprises the heavy chain of isatuximab, which is linked or fused (as further described herein) to a C1q binder with reduced affinity for C1q (as further described herein); as well as to an antibody or antibody construct that comprises two such heavy chains (and two suitable light chains, that are preferably each the light chain of isatuximab). In another embodiment, the invention relates to a polypeptide that comprises the light chain of isatuximab, which is linked or fused (as further described herein) to a C1q binder with reduced affinity for C1q (as further described herein); as well as to an antibody or antibody construct that comprises two such light chains (and two suitable heavy chains, that are preferably each the heavy chain of isatuximab).

[0203] In other embodiments, the anti-CD38 antibody is the Genmab CD38-targeting antibody known as “Clone 003” (see also WO2006 / 099875). The heavy chain of clone 003 is provided below: QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAFSWVRQAPGQGLEWMGRVI PFLGIANSAQKFQGRVTITADKSTSTAYMDLSSLRSEDTAVYYCARDDIAAL GPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK PSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 102)

[0204] The light chain of clone 003 is provided below: DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAAS SLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPRTFGQGTKV EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC (SEQ ID NO: 103)

[0205] In some embodiments, the invention relates to an antibody of the invention (as further described herein) that is directed against CD38 in which the two heavy chains that are present in said antibody of the invention are both the heavy chain of clone 003 and the two light chains that are present in said antibody of the invention are both the light chain of clone 003 (and which antibody of the invention also contains two C1q binders with reduced affinity for C1q, as further described herein).

[0206] In another embodiment, the invention relates to a polypeptide that comprises the heavy chain of clone 003, which is linked or fused (as further described herein) to a C1q binder withreduced affinity for C1q (as further described herein); as well as to an antibody or antibody construct that comprises two such heavy chains (and two suitable light chains, that are preferably each the light chain of clone 003). In another embodiment, the invention relates to a polypeptide that comprises the light chain of clone 003, which is linked or fused (as further described herein) to a C1q binder with reduced affinity for C1q (as further described herein); as well as to an antibody or antibody construct that comprises two such light chains (and two suitable heavy chains, that are preferably each the heavy chain of clone 003).

[0207] In other embodiments, the anti-CD38 antibody is the Morphosys CD38-targeting antibody known as “Moro 3080”. The heavy chain of Moro 3080 is provided below: QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVSNIY SDGSNTFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARNMYRWP FHYFFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFP EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 104)

[0208] The light chain of Moro 3080 is provided below: DIELTQPPSVSVAPGQTARISCSGDNIGNKYVSWYQQKPGQAPVVVIYGDNN RPSGIPERFSGSNSGNTATLTISGTQAEDEADYYCSSYDSSYFVFGGGTKLT VLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVK AGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAP TECS (SEQ ID NO: 105)

[0209] In some embodiments, the invention relates to an antibody of the invention (as further described herein) that is directed against CD38 in which the two heavy chains that are present in said antibody of the invention are both the heavy chain of Moro 3080 and the two light chains that are present in said antibody of the invention are both the light chain of Moro 3080 (and which antibody of the invention also contains two C1q binders with reduced affinity for C1q, as further described herein).

[0210] In another embodiment, the invention relates to a polypeptide that comprises the heavy chain of Moro 3080, which is linked or fused (as further described herein) to a C1q binder with reduced affinity for C1q (as further described herein); as well as to an antibody or antibody construct that comprises two such heavy chains (and two suitable light chains, that are preferably each the light chain of Moro 3080). In another embodiment, the invention relates to a polypeptide that comprises the light chain of Moro 3080, which is linked or fused (as further described herein) to a C1q binder with reduced affinity for C1q (as further described herein); as well as to an antibodyor antibody construct that comprises two such light chains (and two suitable heavy chains, that are preferably each the heavy chain of Moro 3080).

[0211] In other embodiments, the anti-CD38 antibody is the Morphosys CD38-targeting antibody known as “Moro 3087”. The heavy chain of Moro 3087 is provided below: QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYYMNWVRQAPGKGLEWVSGIS GDPSNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLPLVY TGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK PSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:230)

[0212] The light chain of Moro 3087 is provided below: DIELTQPPSVSVAPGQTARISCSGDNLRHYYVYWYQQKPGQAPVLVIYGDSK RPSGIPERFSGSNSGNTATLTISGTQAEDEADYYCQTYTGGASLVFGGGTKV EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC (SEQ ID NO:229)

[0213] In some embodiments, the invention relates to an antibody of the invention (as further described herein) that is directed against CD38 in which the two heavy chains that are present in said antibody of the invention are both the heavy chain of Moro 3087 and the two light chains that are present in said antibody of the invention are both the light chain of Moro 3087 (and which antibody of the invention also contains two C1q binders with reduced affinity for C1q, as further described herein).

[0214] In another embodiment, the invention relates to a polypeptide that comprises the heavy chain of Moro 3087, which is linked or fused (as further described herein) to a C1q binder with reduced affinity for C1q (as further described herein); as well as to an antibody or antibody construct that comprises two such heavy chains (and two suitable light chains, that are preferably each the light chain of Moro 3087). In another embodiment, the invention relates to a polypeptide that comprises the light chain of Moro 3087, which is linked or fused (as further described herein) to a C1q binder with reduced affinity for C1q (as further described herein); as well as to an antibody or antibody construct that comprises two such light chains (and two suitable heavy chains, that are preferably each the heavy chain of Moro 3087).

[0215] In other embodiments, the anti-CD38 antibody is the Morphosys CD38-targeting antibody known as “Moro 3088”. The heavy chain of Moro 3088 is provided below:QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVSGIS SWGSSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREDGSYM TDYFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH KPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRT PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:236)

[0216] The light chain of Moro 3088 is provided below: DIELTQPPSVSVAPGQTARISCSGDNIGHYYVSWYQQKPGQAPVLVIYSDSN RPSGIPERFSGSNSGNTATLTISGTQAEDEADYYCQSYNGTYVFGGGTKVEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC (SEQ ID: 235)

[0217] In some embodiments, the invention relates to an antibody of the invention (as further described herein) that is directed against CD38 in which the two heavy chains that are present in said antibody of the invention are both the heavy chain of Moro 3088 and the two light chains that are present in said antibody of the invention are both the light chain of Moro 3088 (and which antibody of the invention also contains two C1q binders with reduced affinity for C1q, as further described herein).

[0218] In another embodiment, the invention relates to a polypeptide that comprises the heavy chain of Moro 3088, which is linked or fused (as further described herein) to a C1q binder with reduced affinity for C1q (as further described herein); as well as to an antibody or antibody construct that comprises two such heavy chains (and two suitable light chains, that are preferably each the light chain of Moro 3088). In another embodiment, the invention relates to a polypeptide that comprises the light chain of Moro 3088, which is linked or fused (as further described herein) to a C1q binder with reduced affinity for C1q (as further described herein); as well as to an antibody or antibody construct that comprises two such light chains (and two suitable heavy chains, that are preferably each the heavy chain of Moro 3088).

[0219] In other embodiments, the anti-CD38 antibody is mezagitumab. The heavy chain of mezagitumab is provided below: EVQLLESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSDIS WNGGKTHYVDSVKGQFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGSLFHD SSGFYFGHWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV NHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO:242)

[0220] The light chain of mezagitumab is provided below: QSVLTQPPSASGTPGQRVTISCSGSSSNIGDNYVSWYQQLPGTAPKLLIYRD SQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCQSYDSSLSGSVFGGG TKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV TKSFNRGEC(SEQ ID: 241)

[0221] In some embodiments, the invention relates to an antibody of the invention (as further described herein) that is directed against CD38 in which the two heavy chains that are present in said antibody of the invention are both the heavy chain of mezagitumab and the two light chains that are present in said antibody of the invention are both the light chain of mezagitumab (and which antibody of the invention also contains two C1q binders with reduced affinity for C1q, as further described herein).

[0222] In another embodiment, the invention relates to a polypeptide that comprises the heavy chain of mezagitumab, which is linked or fused (as further described herein) to a C1q binder with reduced affinity for C1q (as further described herein); as well as to an antibody or antibody construct that comprises two such heavy chains (and two suitable light chains, that are preferably each the light chain of mezagitumab). In another embodiment, the invention relates to a polypeptide that comprises the light chain of mezagitumab, which is linked or fused (as further described herein) to a C1q binder with reduced affinity for C1q (as further described herein); as well as to an antibody or antibody construct that comprises two such light chains (and two suitable heavy chains, that are preferably each the heavy chain of mezagitumab).

[0223] In other embodiments, the anti-CD38 antibody is the Genmab CD38-targeting antibody known as “Clone 024” (see also WO2006 / 099875). The heavy chain of clone 024 is provided below: EVQLVQSGAEVKKPGESLKISCKGSGYSFSNYWIGWVRQMPGKGLEWMGIIY PHDSDARYSPSFQGQVTFSADKSISTAYLQWSSLKASDTAMYYCARHVGWGS RYWYFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFP EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 246)

[0224] The light chain of clone 024 is provided below: EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPGLLIYDAS NRASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC (SEQ ID NO: 250)

[0225] In some embodiments, the invention relates to an antibody of the invention (as further described herein) that is directed against CD38 in which the two heavy chains that are present in said antibody of the invention are both the heavy chain of clone 024 and the two light chains that are present in said antibody of the invention are both the light chain of clone 024 (and which antibody of the invention also contains two C1q binders with reduced affinity for C1q, as further described herein).

[0226] In another embodiment, the invention relates to a polypeptide that comprises the heavy chain of clone 024, which is linked or fused (as further described herein) to a C1q binder with reduced affinity for C1q (as further described herein); as well as to an antibody or antibody construct that comprises two such heavy chains (and two suitable light chains, that are preferably each the light chain of clone 024). In another embodiment, the invention relates to a polypeptide that comprises the light chain of clone 024, which is linked or fused (as further described herein) to a C1q binder with reduced affinity for C1q (as further described herein); as well as to an antibody or antibody construct that comprises two such light chains (and two suitable heavy chains, that are preferably each the heavy chain of clone 024).

[0227] In some embodiments, the anti-CD38 antibody or fragment thereof is a single domain antibody such as MU1053. The amino acid sequence of MU1053 is provided below: QVQLQESGGGLVQAGGSLRLSCTGSGRTFRNYPMAWFRQAPGKEREFVAGITWVGAST LYADFAKGRFTISRDNAKNTVYLQMNSLKPEDTAVYSCAAGRGIVAGRIPAEYADWGQ GTQVTVSS (SEQ ID NO: 77)

[0228] In other embodiments, the anti-CD38 antibody is the anti-CD38 antibody CM313. The heavy chain of CM313 is provided below: EVQLKQSGPGLMQPSQSLSITCTVSGFSLTSYGIHWLRQSPGKGLEWLGV IWRGGSTDYNAAFMSRLSITKDNSKSQVFFKMNSLQGDDTAIYYCAKGKV TTGFYFDFWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY ICNVNHKPSNTKVDKKAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 264)

[0229] The light chain of CM313 is provided below: DIQMTQSSSSFSVSLGDRVTITCKASEDIYNRLVWYQQKPGNAPGLLISGVTSLETGVPSRFSGSGSGKDYTLTITSLQTEDVATYYCQQYWSTPYTFGG GTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC (SEQ ID NO: 265)

[0230] In another aspect,, the invention relates to an antibody of the invention against CD38 in which the two heavy chains that are present in said antibody of the invention are both the heavy chain of CM313 and the two light chains that are present in said antibody of the invention are both the light chain of CM313 (and which antibody of the invention also contains two C1q binders, as further described herein).

[0231] In another embodiment, the invention relates to a polypeptide that comprises the heavy chain of CM313, which is linked or fused (as further described herein) to a C1q binder (as further described herein); as well as to an antibody or antibody construct that comprises two such heavy chains (and two suitable light chains, that are preferably each the light chain of CM313). In another embodiment, the invention relates to a polypeptide that comprises the light chain of CM313, which is linked or fused (as further described herein) to a C1q binder (as further described herein); as well as to an antibody or antibody construct that comprises two such light chains (and two suitable heavy chains, that are preferably each the heavy chain of CM313). Reference is also made to Example 7 and FIG.29.

[0232] In some embodiments, the anti-RANKL antibody is denosumab. The heavy chain of denosumab is provided below: EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGIT GSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDPGTTV IMSWFDPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFP EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 78)

[0233] The light chain of denosumab is provided below: EIVLTQSPGTLSLSPGERATLSCRASQSVRGRYLAWYQQKPGQAPRLLIYGA SSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVFYCQQYGSSPRTFGQGTK VEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC (SEQ ID NO: 79)

[0234] In some embodiments, the invention relates to an antibody of the invention (as further described herein) that is directed against RANK-L. In particular, the invention relates to such an antibody of the invention in which the two heavy chains that are present in said antibody of theinvention are both the heavy chain of denosumab and the two light chains that are present in said antibody of the invention are both the light chain of denosumab (and which antibody of the invention also contains two C1q binders with reduced affinity for C1q, as further described herein).

[0235] In another embodiment, the invention relates to a polypeptide that comprises the heavy chain of denosumab, which is linked or fused (as further described herein) to a C1q binder with reduced affinity for C1q (as further described herein); as well as to an antibody or antibody construct that comprises two such heavy chains (and two suitable light chains, that are preferably each the light chain of denosumab). In another embodiment, the invention relates to a polypeptide that comprises the light chain of denosumab, which is linked or fused (as further described herein) to a C1q binder with reduced affinity for C1q (as further described herein); as well as to an antibody or antibody construct that comprises two such light chains (and two suitable heavy chains, that are preferably each the heavy chain of denosumab).

[0236] In some embodiments, the anti-IL-4 antibody is duplimab. The heavy chain of duplimab is provided below: EVQLVESGGGLEQPGGSLRLSCAGSGFTFRDYAMTWVRQAPGKGLEWVSSIS GSGGNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDRLSIT IRPRYYGLDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTC NVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISR TPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLT VDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO: 80)

[0237] The light chain of duplimab is provided below: DIVMTQSPLSLPVTPGEPASISCRSSQSLLYSIGYNYLDWYLQKSGQSPQLL IYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGFYYCMQALQTPYTFG QGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC (SEQ ID NO: 81)

[0238] In some embodiments, the invention relates to an antibody of the invention (as further described herein) that is directed against IL-4. In particular, the invention relates to such an antibody of the invention in which the two heavy chains that are present in said antibody of the invention are both the heavy chain of duplimab and the two light chains that are present in said antibody of the invention are both the light chain of duplimab (and which antibody of the invention also contains two C1q binders with reduced affinity for C1q, as further described herein).

[0239] In another embodiment, the invention relates to a polypeptide that comprises the heavy chain of duplimab, which is linked or fused (as further described herein) to a C1q binder withreduced affinity for C1q (as further described herein); as well as to an antibody or antibody construct that comprises two such heavy chains (and two suitable light chains, that are preferably each the light chain of duplimab). In another embodiment, the invention relates to a polypeptide that comprises the light chain of duplimab, which is linked or fused (as further described herein) to a C1q binder with reduced affinity for C1q (as further described herein); as well as to an antibody or antibody construct that comprises two such light chains (and two suitable heavy chains, that are preferably each the heavy chain of duplimab).

[0240] In some embodiments, the anti-CD33 antibody is gemtuzumab ozogamicin. The heavy chain of gemtuzumab ozogamicin is provided below: EVQLVQSGAEVKKPGSSVKVSCKASGYTITDSNIHWVRQAPGQSLEWIGYIY PYNGGTDYNQKFKNRATLTVDNPTNTAYMELSSLRSEDTAFYYCVNGNPWLA YWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNT KVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEG NVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 82)

[0241] The light chain of gemtuzumab ozogamicin is provided below: DIQLTQSPSTLSASVGDRVTITCRASESLDNYGIRFLTWFQQKPGKAPKLLM YAASNQGSGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQTKEVPWSFGQ GTKVEVKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDN ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC (SEQ ID NO: 83)

[0242] In some embodiments, the invention relates to an antibody of the invention (as further described herein) that is directed against CD33. In particular, the invention relates to such an antibody of the invention in which the two heavy chains that are present in said antibody of the invention are both the heavy chain of gemtuzumab ozogamicin and the two light chains that are present in said antibody of the invention are both the light chain of gemtuzumab ozogamicin (and which antibody of the invention also contains two C1q binders with reduced affinity for C1q, as further described herein).

[0243] In another embodiment, the invention relates to a polypeptide that comprises the heavy chain of gemtuzumab ozogamicin, which is linked or fused (as further described herein) to a C1q binder with reduced affinity for C1q (as further described herein); as well as to an antibody or antibody construct that comprises two such heavy chains (and two suitable light chains, that are preferably each the light chain of gemtuzumab ozogamicin). In another embodiment, the invention relates to a polypeptide that comprises the light chain of gemtuzumab ozogamicin, which is linkedor fused (as further described herein) to a C1q binder with reduced affinity for C1q (as further described herein); as well as to an antibody or antibody construct that comprises two such light chains (and two suitable heavy chains, that are preferably each the heavy chain of gemtuzumab ozogamicin).

[0244] In some embodiments, the anti-CTLA-4 antibody is ipilimumab. The heavy chain of ipilimumab is provided below: QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKGLEWVTFIS YDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGP FDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS NTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEV TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 84)

[0245] The light chain of ipilimumab is provided below: EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQAPRLLIYGA FSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTK VEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC (SEQ ID NO: 85)

[0246] In some embodiments, the invention relates to an antibody of the invention (as further described herein) that is directed against CTLA-4. In particular, the invention relates to such an antibody of the invention in which the two heavy chains that are present in said antibody of the invention are both the heavy chain of ipilimumab and the two light chains that are present in said antibody of the invention are both the light chain of ipilimumab (and which antibody of the invention also contains two C1q binders with reduced affinity for C1q, as further described herein).

[0247] In another embodiment, the invention relates to a polypeptide that comprises the heavy chain of ipilimumab, which is linked or fused (as further described herein) to a C1q binder with reduced affinity for C1q (as further described herein); as well as to an antibody or antibody construct that comprises two such heavy chains (and two suitable light chains, that are preferably each the light chain of ipilimumab). In another embodiment, the invention relates to a polypeptide that comprises the light chain of ipilimumab, which is linked or fused (as further described herein) to a C1q binder with reduced affinity for C1q (as further described herein); as well as to an antibody or antibody construct that comprises two such light chains (and two suitable heavy chains, that are preferably each the heavy chain of ipilimumab).

[0248] In some embodiments, the anti-CTLA-4 antibody is tremelimumab. The heavy chain of tremelimumab is provided below: QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKGLEWVTFIS YDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGP FDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS NTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEV TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 86)

[0249] The light chain of tremelimumab is provided below: EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQAPRLLIYGA FSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTK VEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC (SEQ ID NO: 87)

[0250] In some embodiments, the invention relates to an antibody of the invention (as further described herein) that is directed against CTLA-4 in which the two heavy chains that are present in said antibody of the invention are both the heavy chain of tremelimumab and the two light chains that are present in said antibody of the invention are both the light chain of tremelimumab (and which antibody of the invention also contains two C1q binders with reduced affinity for C1q, as further described herein).

[0251] In another embodiment, the invention relates to a polypeptide that comprises the heavy chain of tremelimumab, which is linked or fused (as further described herein) to a C1q binder with reduced affinity for C1q (as further described herein); as well as to an antibody or antibody construct that comprises two such heavy chains (and two suitable light chains, that are preferably each the light chain of tremelimumab). In another embodiment, the invention relates to a polypeptide that comprises the light chain of tremelimumab, which is linked or fused (as further described herein) to a C1q binder with reduced affinity for C1q (as further described herein); as well as to an antibody or antibody construct that comprises two such light chains (and two suitable heavy chains, that are preferably each the heavy chain of tremelimumab).

[0252] In some embodiments, the anti-integrin antibody is natalizumab. The heavy chain of natalizumab is provided below: QVQLVQSGAEVKKPGASVKVSCKASGFNIKDTYIHWVRQAPGQRLEWMGRID PANGYTKYDPKFQGRVTITADTSASTAYMELSSLRSEDTAVYYCAREGYYGN YGVYAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVD KSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 88)

[0253] The light chain of natalizumab is provided below: DIQMTQSPSSLSASVGDRVTITCKTSQDINKYMAWYQQTPGKAPRLLIHYTS ALQPGIPSRFSGSGSGRDYTFTISSLQPEDIATYYCLQYDNLWTFGQGTKVE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC (SEQ ID NO: 89)

[0254] In some embodiments, the invention relates to an antibody of the invention (as further described herein) that is directed against an integrin (and in particular, against integrin alpha-4). In particular, the invention relates to such an antibody of the invention in which the two heavy chains that are present in said antibody of the invention are both the heavy chain of natalizumab and the two light chains that are present in said antibody of the invention are both the light chain of natalizumab (and which antibody of the invention also contains two C1q binders with reduced affinity for C1q, as further described herein).

[0255] In another embodiment, the invention relates to a polypeptide that comprises the heavy chain of natalizumab, which is linked or fused (as further described herein) to a C1q binder with reduced affinity for C1q (as further described herein); as well as to an antibody or antibody construct that comprises two such heavy chains (and two suitable light chains, that are preferably each the light chain of natalizumab). In another embodiment, the invention relates to a polypeptide that comprises the light chain of natalizumab, which is linked or fused (as further described herein) to a C1q binder with reduced affinity for C1q (as further described herein); as well as to an antibody or antibody construct that comprises two such light chains (and two suitable heavy chains, that are preferably each the heavy chain of natalizumab).

[0256] In some embodiments, the anti-PD-1 antibody is nivolumab. The heavy chain of nivolumab is SEQ ID NO: 100. QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIW YDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWG QGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVD KRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS QEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVF SCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 100)

[0257] The light chain of nivolumab is provided below in SEQ ID NO: 101:EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDAS NRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKV EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC (SEQ ID NO: 101)

[0258] In some embodiments, the invention relates to an antibody of the invention (as further described herein) that is directed against PD-1. In particular, the invention relates to such an antibody of the invention in which the two heavy chains that are present in said antibody of the invention are both the heavy chain of nivolumab and the two light chains that are present in said antibody of the invention are both the light chain of nivolumab (and which antibody of the invention also contains two C1q binders with reduced affinity for C1q, as further described herein).

[0259] In another embodiment, the invention relates to a polypeptide that comprises the heavy chain of nivolumab, which is linked or fused (as further described herein) to a C1q binder with reduced affinity for C1q (as further described herein); as well as to an antibody or antibody construct that comprises two such heavy chains (and two suitable light chains, that are preferably each the light chain of nivolumab). In another embodiment, the invention relates to a polypeptide that comprises the light chain of nivolumab, which is linked or fused (as further described herein) to a C1q binder with reduced affinity for C1q (as further described herein); as well as to an antibody or antibody construct that comprises two such light chains (and two suitable heavy chains, that are preferably each the heavy chain of nivolumab).

[0260] In other embodiments, the anti-PD-1 antibody is pembrolizumab. The heavy chain of pembrolizumab is provided below: QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGIN PSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFD MGFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHK PSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLS (SEQ ID NO: 90)

[0261] The light chain of pembrolizumab is provided below: EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLI YLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGG GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDN ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC (SEQ ID NO: 91)

[0262] In some embodiments, the invention relates to an antibody of the invention (as further described herein) that is directed against PD-1 in which the two heavy chains that are present insaid antibody of the invention are both the heavy chain of pembrolizumab and the two light chains that are present in said antibody of the invention are both the light chain of pembrolizumab (and which antibody of the invention also contains two C1q binders with reduced affinity for C1q, as further described herein).

[0263] In another embodiment, the invention relates to a polypeptide that comprises the heavy chain of pembrolizumab, which is linked or fused (as further described herein) to a C1q binder with reduced affinity for C1q (as further described herein); as well as to an antibody or antibody construct that comprises two such heavy chains (and two suitable light chains, that are preferably each the light chain of pembrolizumab). In another embodiment, the invention relates to a polypeptide that comprises the light chain of pembrolizumab, which is linked or fused (as further described herein) to a C1q binder with reduced affinity for C1q (as further described herein); as well as to an antibody or antibody construct that comprises two such light chains (and two suitable heavy chains, that are preferably each the heavy chain of pembrolizumab).

[0264] In some embodiments, the anti-CD20 antibody is rituximab. The heavy chain of rituximab is provided below: QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIY PGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGG DWYFNVWGAGTTVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH KPSNTKVDKKAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRT PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 92)

[0265] The light chain of rituximab is provided below: QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSN LASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC (SEQ ID NO: 93)

[0266] In some embodiments, the invention relates to an antibody of the invention (as further described herein) that is directed against CD20. In particular, the invention relates to such an antibody of the invention in which the two heavy chains that are present in said antibody of the invention are both the heavy chain of rituximab and the two light chains that are present in said antibody of the invention are both the light chain of rituximab (and which antibody of the invention also contains two C1q binders with reduced affinity for C1q, as further described herein).

[0267] In another embodiment, the invention relates to a polypeptide that comprises the heavy chain of rituximab, which is linked or fused (as further described herein) to a C1q binder with reduced affinity for C1q (as further described herein); as well as to an antibody or antibody construct that comprises two such heavy chains (and two suitable light chains, that are preferably each the light chain of rituximab). In another embodiment, the invention relates to a polypeptide that comprises the light chain of rituximab, which is linked or fused (as further described herein) to a C1q binder with reduced affinity for C1q (as further described herein); as well as to an antibody or antibody construct that comprises two such light chains (and two suitable heavy chains, that are preferably each the heavy chain of rituximab).

[0268] In some embodiments, the anti-CD20 antibody is 11B8. The heavy chain of 11B8 is given below: EVQLVQSGGGLVHPGGSLRLSCTGSGFTFSYHAMHWVRQAPGKGLEWVSIIGTGGVT YYADSVKGRFTISRDNVKNSLYLQMNSLRAEDMAVYYCARDYYGAGSFYDGLYGM DVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:106)

[0269] The light chain of 11B8 is given below: EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPA RFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSDWPLTFGGGTKVEIKRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 107).

[0270] In some embodiments, the invention relates to an antibody of the invention (as further described herein) that is directed against CD20 in which the two heavy chains that are present in said antibody of the invention are both the heavy chain of 11B8 and the two light chains that are present in said antibody of the invention are both the light chain of 11B8 (and which antibody ofthe invention also contains two C1q binders with reduced affinity for C1q, as further described herein).

[0271] In another embodiment, the invention relates to a polypeptide that comprises the heavy chain of 11B8, which is linked or fused (as further described herein) to a C1q binder with reduced affinity for C1q (as further described herein); as well as to an antibody or antibody construct that comprises two such heavy chains (and two suitable light chains, that are preferably each the light chain of 11B8). In another embodiment, the invention relates to a polypeptide that comprises the light chain of 11B8, which is linked or fused (as further described herein) to a C1q binder with reduced affinity for C1q (as further described herein); as well as to an antibody or antibody construct that comprises two such light chains (and two suitable heavy chains, that are preferably each the heavy chain of 11B8).

[0272] In some embodiments, the anti-CD137 antibody is urelumab. The heavy chain of urelumab is provided below: QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQSPEKGLEWIGEIN HGGYVTYNPSLESRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDYGPGNY DWYFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH KPSNTKVDKKVEPKSCAAAHHHHHHHH (SEQ ID NO: 94) or QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQSPEKGLEWIGEIN HGGYVTYNPSLESRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDYGPGNY DWYFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH KPSNTKVDKKVEPKSCAAA (SEQ ID NO: 293)

[0273] The light chain of urelumab is provided below: EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDAS NRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPALTFGGGT KVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT KSFNRGEC (SEQ ID NO: 95)

[0274] In some embodiments, the invention relates to an antibody of the invention (as further described herein) that is directed against CD137. In particular, the invention relates to such an antibody of the invention in which the two heavy chains that are present in said antibody of the invention are both the heavy chain of urelumab and the two light chains that are present in said antibody of the invention are both the light chain of urelumab (and which antibody of the invention also contains two C1q binders with reduced affinity for C1q, as further described herein).

[0275] In another embodiment, the invention relates to a polypeptide that comprises the heavy chain of urelumab, which is linked or fused (as further described herein) to a C1q binder with reduced affinity for C1q (as further described herein); as well as to an antibody or antibody construct that comprises two such heavy chains (and two suitable light chains, that are preferably each the light chain of urelumab). In another embodiment, the invention relates to a polypeptide that comprises the light chain of urelumab, which is linked or fused (as further described herein) to a C1q binder with reduced affinity for C1q (as further described herein); as well as to an antibody or antibody construct that comprises two such light chains (and two suitable heavy chains, that are preferably each the heavy chain of urelumab).

[0276] In some embodiments, the anti-IL12 / IL23 antibody is ustekinumab. The heavy chain of ustekinumab is provided below: EVQLVQSGAEVKKPGESLKISCKGSGYSFTTYWLGWVRQMPGKGLDWIGIMS PVDSDIRYSPSFQGQVTMSVDKSITTAYLQWNSLKASDTAMYYCARRRPGQG YFDFWGQGTLVTVSSSSTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP SNTKVDKRVEPKSCDKTH (SEQ ID NO: 96)

[0277] The light chain of ustekinumab is provided below: DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAAS SLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNIYPYTFGQGTKL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC (SEQ ID NO: 97)

[0278] In some embodiments, the invention relates to an antibody of the invention (as further described herein) that is directed against. In particular, the invention relates to such an antibody of the invention in which the two heavy chains that are present in said antibody of the invention are both the heavy chain of ustekinumab and the two light chains that are present in said antibody of the invention are both the light chain of ustekinumab (and which antibody of the invention also contains two C1q binders with reduced affinity for C1q, as further described herein).

[0279] In another embodiment, the invention relates to a polypeptide that comprises the heavy chain of ustekinumab, which is linked or fused (as further described herein) to a C1q binder with reduced affinity for C1q (as further described herein); as well as to an antibody or antibody construct that comprises two such heavy chains (and two suitable light chains, that are preferably each the light chain of ustekinumab). In another embodiment, the invention relates to a polypeptide that comprises the light chain of ustekinumab, which is linked or fused (as further described herein) to a C1q binder with reduced affinity for C1q (as further described herein); as well as to an antibodyor antibody construct that comprises two such light chains (and two suitable heavy chains, that are preferably each the heavy chain of ustekinumab).

[0280] In some embodiments, the anti-CD19 antibody is tafasitamab (SEQ ID NO: 98 and SEQ ID NO: 99). The heavy chain of tafasitamab is provided below in SEQ ID NO: 98. EVQLVESGGGLVKPGGSLKLSCAASGYTFTSYVMHWVRQAPGKGLEWIGYIN PYNDGTKYNEKFQGRVTISSDKSISTAYMELSSLRSEDTAMYYCARGTYYYG TRVFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH KPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRT PEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTV VHQDWLNGKEYKCKVSNKALPAPEEKTISKTKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 98)

[0281] The light chain of tafasitamab is provided below in SEQ ID NO: 99: DIVMTQSPATLSLSPGERATLSCRSSKSLQNVNGNTYLYWFQQKPGQSPQL LIYRMSNLNSGVPDRFSGSGSGTEFTLTISSLEPEDFAVYYCMQHLEYPIT FGAGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC (SEQ ID NO: 99)

[0282] In some embodiments, the invention relates to an antibody of the invention (as further described herein) that is directed against CD19. In particular, the invention relates to such an antibody of the invention in which the two heavy chains that are present in said antibody of the invention are both the heavy chain of tafasitamab and the two light chains that are present in said antibody of the invention are both the light chain of tafasitamab (and which antibody of the invention also contains two C1q binders with reduced affinity for C1q, as further described herein).

[0283] In another embodiment, the invention relates to a polypeptide that comprises the heavy chain of tafasitamab, which is linked or fused (as further described herein) to a C1q binder with reduced affinity for C1q (as further described herein); as well as to an antibody or antibody construct that comprises two such heavy chains (and two suitable light chains, that are preferably each the light chain of tafasitamab). In another embodiment, the invention relates to a polypeptide that comprises the light chain of tafasitamab, which is linked or fused (as further described herein) to a C1q binder with reduced affinity for C1q (as further described herein); as well as to an antibody or antibody construct that comprises two such light chains (and two suitable heavy chains, that are preferably each the heavy chain of tafasitamab). Pharmaceutical Compositions

[0284] One aspect of the present disclosure relates to a composition comprising one or more of the bispecific constructs provided herein. In some embodiments, the composition is apharmaceutical composition. A pharmaceutical composition is a composition comprising one or more substances that have medicinal properties, together with a pharmaceutically acceptable diluent, carrier, or excipient described herein. Details of pharmaceutical compositions are provided herein below.

[0285] The bispecific constructs can be formulated as pharmaceutical compositions and administered to a mammalian host, such as a human patient in a variety of forms adapted to the chosen route of administration. Routes for administration include, for example, intravenous, intra- arterial, subcutaneous, intramuscular, intraperitoneal, intravitreal and other routes selected by one of skill in the art. Administration can also be achieved by nebulization. In another approach, the bispecific constructs can be administered as DNA by AAV and then expressed from the vector Administration forms are described elsewhere herein.

[0286] In some embodiments, the solutions of the bispecific constructs can for example be prepared in water or saline, and optionally mixed with a nontoxic surfactant. In some embodiments, the pharmaceutical compositions as described herein are formulated for intravenous or intra-arterial administration may include sterile aqueous solutions that may also contain buffers, liposomes, diluents and other suitable additives. In some embodiments, the pharmaceutical composition can also comprise or include serum.

[0287] In some embodiments, the pharmaceutical composition is in a dosage form. In some embodiments, the dosage forms are suitable for injection or infusion can include sterile aqueous solutions or dispersions comprising the bispecific constructs that are adapted for administration by encapsulation in liposomes. The ultimate dosage form must be sterile, fluid and stable under the conditions of manufacture and storage.

[0288] Sterile injectable solutions are prepared by incorporating the bispecific constructs in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization. Methods of Treating a Disease

[0289] The bispecific constructs provided herein can be used for both in vivo and in vitro methods as well as medical and non-medical procedures. In some embodiments, the bispecific constructs provided herein are provided for use as a medicament. In certain embodiments, the bispecific constructs and compositions are provided for treatment of a disorder, a clinical or physiological condition associated with complement activity. The bispecific constructs and compositions can also be applied for use in the preparation of a medicament, for example for the treatment of a disease or a disorder, a clinical or physiological condition associated with complement activity.

[0290] In some embodiments, the bispecific constructs as disclosed herein comprise an antigen- binding moiety that binds to a target antigen that is a protein implicated in a disease. Accordingly, the bispecific constructs may be used in the treatment of the disease in which the target protein is implicated. In some embodiments, the disease is a cancer, an inflammatory disease, an autoimmune disease, an infectious disease, a cardiac disease, a genetic disease or a neurological disease. In some embodiments, the disease is a is a cancer, an autoimmune disease, an inflammatory disease, or a neurological disease.

[0291] In certain embodiments, the bispecific constructs as disclosed herein comprise an antigen- binding moiety that binds to a target antigen that is a protein implicated in a cancer. Accordingly, the bispecific constructs may be used in the treatment of cancer, such as in the treatment of the cancer in which the target protein is implicated. The term “cancer” as used herein refers to proliferative diseases, such as lymphomas, carcinoma, lymphoma, blastoma, sarcoma, leukemia, lymphocytic leukemias, lung cancer, non-small cell lung (NSCL) cancer, bone cancer, bronchoalveolar cell lung cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, gastric cancer, colorectal cancer (CRC), pancreatic cancer, breast cancer, triple-negative breast cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, prostate cancer, cancer of the bladder, cancer of the kidney or ureter, renal cell carcinoma, carcinoma of the renal pelvis, mesothelioma, hepatocellular cancer, biliary cancer, neoplasms of the central nervous system (CNS), spinal axis tumors, brain stem glioma, glioblastoma multiforme, astrocytomas, schwanomas, ependymonas, medulloblastomas, meningiomas, squamous cell carcinomas, pituitary adenoma and Ewings sarcoma, melanoma, multiple myeloma, B-cell cancer (lymphoma), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia, including refractory versions of any of the above cancers, or a combination of one or more of the above cancers. In some embodiments, the cancer is a breast cancer, a colorectal cancer, a lung cancer, a leukemia, a bladder cancer, a lymphoma, a melanoma, a carcinoma, a kidney cancer, a prostate cancer, a bone cancer, a brain tumor, an adenocarcinoma, an adrenal cancer, a bile duct cancer, a cervical cancer, a pancreatic cancer, a thyroid cancer, an appendix cancer, a myeloma, a sarcoma, a cancer that primarily affects minors or children, or any combination thereof.

[0292] In some embodiments, the bispecific constructs as disclosed herein comprise an antigen- binding moiety that binds to a target antigen that is a protein implicated in an inflammatory disease. Accordingly, the bispecific constructs may be used in the treatment of inflammatory disease, such as in the treatment of the inflammatory disease in which the target protein is implicated. In some embodiments, the inflammatory disease can include but is not limited to fatty liver disease, endometriosis, type 1 diabetes mellitus, type 2 diabetes mellitus, asthma, obesity, inflammatory bowel disease, rheumatoid arthritis, colitis, gout, sinusitis, vasculitis, ankylosing spondylitis, or an autoimmune disease. In other embodiments, the bispecific constructs as disclosed herein comprise an antigen-binding moiety that binds to a target protein implicated in an inflammatory disease that is an autoimmune disease. The term “autoimmune disease” as used herein refers to a disease that results when a subject’s immune system is overactive causing it to attack and damage tissues in the subject’s body. In some embodiments, the autoimmune disease can include but is not limited to lupus, rheumatoid arthritis, type 1 diabetes, multiple sclerosis, psoriasis, Grave’s disease, inflammatory bowel disease, scleroderma, Hashimoto thyroiditis, Sjögren syndrome, Celiac disease, Myasthenia gravis, Addison's disease, alopecia areata, autoimmune angioedema, or vitiligo. In some embodiments, the autoimmune disease is selected from multiple sclerosis, psoriasis, psoriatic arthritis, rheumatoid arthritis, systemic lupus erythematosus, Crohn's disease, Sjogren's syndrome, Behcet's disease, ulcerative colitis, and Guillain-Barre syndrome.

[0293] In some embodiments, the bispecific constructs as disclosed herein comprise an antigen- binding moiety that binds to a target antigen that is a protein implicated in a neurological disease. Accordingly, the bispecific constructs may be used in the treatment of neurological disease, such as in the treatment of the neurological disease in which the target protein is implicated. In some embodiments, the neurological disease can include but is not limited to epilepsy, dementia, stroke, headaches, Cerebral palsy, Multiple sclerosis, Amyotrophic lateral sclerosis, Meningitis, acute spinal cord injury, encephalitis, neuronal migration disorder, spina bifida, Barth syndrome, cervical spinal stenosis, Lewy body dementia, or muscular dystrophy.

[0294] In some embodiments, the bispecific constructs as disclosed herein comprise an antigen- binding moiety that binds to a target protein implicated in an infectious disease. Accordingly, the bispecific constructs may be used in the treatment of infectious disease, such as in the treatment of the infectious disease in which the target protein is implicated. For example, in some aspects the infectious disease can include but is not limited to a chronic viral infection like HIV (human immunodeficiency virus), HBV (hepatitis B virus), HCV (hepatitis C), HSV1 (herpes simplex virus type 1), HSV2 (herpes simplex virus type 2), CMV (cytomegalovirus), LCMV (lymphocytic choriomeningitis virus) or EBV (Epstein-Barr virus). In some embodiments, the infectious diseaseis hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, bacterial infections, fungal infections, protozoal infections or parasitic infections.

[0295] In some other embodiments, the bispecific constructs as described herein relates to a method of treating a disorder, a clinical or physiological condition associated with complement regulation, such as complement activation, said method comprising administering a therapeutically effective amount of a bispecific construct or composition as defined herein to a subject in need thereof. The bispecific constructs as described herein can be used alone or coupled to, or combined with, therapeutically useful agents. The bispecific constructs can be administered to mammals suffering from any disease caused by dysregulation or over activation of the complement system or any of the diseases or clinical conditions mentioned herein. Such administration can provide therapeutic and curative treatments, as well as prophylactic, preventative and / or ameliorating measures. Methods of reducing antibody-therapy induced side effects

[0296] In some embodiments, the complement factor-engaging domain can activate human complement system. The complement system is part of the innate immune system and plays an important role in protection against invading microorganisms and in maintenance of homoeostasis. More than 50 proteins and protein fragments make up the complement system, including serum proteins, serosal proteins, and cell membrane bound receptors and regulatory proteins. A subset of the complement proteins circulates as inactive precursors (pro-proteins). When stimulated by one of several triggers, proteases in the system cleave specific proteins to initiate an amplifying cascade of further cleavages. The end-result of this activation cascade includes massive amplification of the response, enhanced phagocytosis and pathogen lysis, clearance of immune complexes and apoptotic cells, inflammation, stimulation of adaptive immune responses and assembly of the cell- killing membrane attack complex. Uncontrolled activation or lack of proper regulation of complement is involved in a range of diseases and the bispecific constructs described herein provides means for pharmacological regulation of the complement cascade in order to ameliorate disease outcome. EXAMPLES

[0297] For a better understanding of the present disclosure and of its many advantages, the following examples are given by way of illustration and without limiting the scope of this disclosure. For further experimental details, reference is also made to the figures and examplesof the International application of applicant / assignee entitled “Improved antibodies and antibody constructs against CD38” with the same filing date as the present application and to the International application of applicant / assignee entitled “Improved antibodies and antibody constructs against CD20” with the same filing date as the present application. EXAMPLE 1: GENERATION BISPECIFIC CONSTRUCTS COMPRISING LOW AFFINITY COMPLEMENT FACTOR-ENGAGING DOMAINS

[0298] This example described production of bispecific constructs of the present disclosure from purified single-domain antibody preparations. Immunization and selection was performed using human C1q. Nanobodies were expressed in E. coli using 2xTY media overnight. Cells were centrifuged for 15 minutes at 4000 g and resuspended in 30 ml of PBS containing 1 mM Benzamidine, and 0.1 mM PMSF. Cells were opened with sonication on wet ice and subsequently centrifuged for 20 minutes at 15,000×g at 4°C. Supernatants were added to a 0.5 ml Nickel NTA (Ni-NTA) slurry and incubated rolling at 4°C for 30 min. Beads were decanted on a gravity column and washed with 2 x 50 ml cold PBS containing 500 sodium chloride (wash buffer). The polypeptide constructs as described herein were then eluted with wash buffer containing 300 mM imidazole at a pH 8.0. Polypeptide purity was evaluated by SDS-PAGE and polypeptide constructs were then dialyzed against PBS overnight at 4°C. Samples were centrifuged for 10 minutes at 4000 rpm at 4°C and the polypeptide constructs was concentrated to approximate 0.5 ml and loaded on a Superdex 75 Increase 10 / 300 GL column in PBS. Eluted fractions containing polypeptide constructs were analyzed by SDS-PAGE and fractions containing the polypeptide constructs were pooled.

[0299] Selected residues with respect to the amino acid sequence of SEQ ID NO: 3 (N31, T52, H58, N73, N100, P101, P102, W104, P105) were mutated to alanine, and the variants were expressed and purified as single domain C1q nanobodies. Single domain antibodies targeting the CD38 having an amino acid sequence of SEQ ID NO: 77 were genetically fused to the purified single domain C1q nanobodies using a 10 amino acid linker (GGGGSGGGGS) (SEQ ID NO: 47). C1q binding kinetics with the globular head of human C1q were determined by Bio-Layer Interferometry (BLI) (FIG.1A - FIG.1J, TABLE 2). TABLE 2: Binding affinities of different exemplary bispecific constructsMutation SEQ ID NO: KD(M) kon(1 / Ms) kdis(1 / s) WT SEQ ID NO: 3 2.69E-09 5.38E+05 1.45E-03 N73A SEQ ID NO: 19 3.63E-09 5.35E+05 1.94E-03 N100A SEQ ID NO: 20 4.07E-09 3.34E+05 1.36E-03 P101A SEQ ID NO: 21 5.83E-09 4.46E+05 2.60E-03 H58A SEQ ID NO: 22 7.85E-09 5.18E+05 4.07E-03 P102A SEQ ID NO: 23 1.28E-08 2.17E+05 2.78E-03 N31A SEQ ID NO: 24 1.83E-08 4.44E+05 8.12E-03 P105A SEQ ID NO: 25 8.71E-07 1.25E+05 1.09E-01 W104A SEQ ID NO: 26 >2E-06 1.14E+05 2.65E-01 T53G SEQ ID NO: 27 >2E-06 1.38E+05 5.44E-01

[0300] Additionally, selected residues with respect to the amino acid sequence of SEQ ID NO: 2 (D101, T102, Y113, E114, or a combination of T102 / Y113, T102 / E114) were mutated to alanine, and the variants were expressed and purified as single domain C1q nanobodies. Single domain antibodies targeting the CD38 having an amino acid sequence of SEQ ID NO: 77 were genetically fused to the purified single domain C1q nanobodies to the purified single domain C1q nanobodies using a polypeptide linker (SEQ ID NO: 46 – SEQ ID NO: 48). C1q binding kinetics with the globular head of human C1q were determined by BLI (TABLE 3). TABLE 3: Binding affinities of different exemplary bispecific constructs Mutation SEQ ID NO: KD(nM) WT SEQ ID NO: 2 2.4 Y113A SEQ ID NO: 4 36.4 E114A SEQ ID NO: 5 154 D101A SEQ ID NO: 6 191 T102A, Y113A SEQ ID NO: 7 1270 T102A, E114A SEQ ID NO: 8 1300

[0301] Furthermore, humanized variants of C1q complement factor-engaging single-domain antibodies were identified and optimized. CDRs from SEQ ID NO: 3 - SEQ ID NO: 4 were grafted into humanized frameworks, expressed and characterized with BLI and thermodynamic integrations (Ti). Exemplary humanized C1q complement factor-engaging single-domain antibodies retained binding affinity to C1q (FIG.3A - FIG.3F, TABLE 4).TABLE 4: Binding affinities of different exemplary bispecific constructs SEQ ID NO: KD(nM) Ratio Ti (°C) SEQ ID NO: 2 1 66.94 SEQ ID NO: 9 NA 66.21 SEQ ID NO: 10 2.41 69.28 SEQ ID NO: 11 11.9 67.65 SEQ ID NO: 12 6.29 61.84 SEQ ID NO: 13 21.2 67.86

[0302] The humanized C1q-binding single domain antibody having an amino acid sequence of SEQ ID NO: 10 was then used to produce bispecific constructs. Alanine mutations in CDR1- CDR3 of the humanized C1q-binding single domain antibody were produced (SEQ ID NO: 14 - SEQ ID NO: 18 and SEQ ID NO: 28 – SEQ ID NO: 42 below) and were linked to the CD38- binding single-domain antibody having an amino acid sequence of SEQ ID NO: 77. The binding affinity for each bispecific construct was then assessed (FIG.4A - FIG.4G, TABLE 5, FIG 7). Compared to the non-humanized wild-type construct of SEQ ID NO: 2, the constructs disclosed in TABLE 5 have approximately 10 times higher KD and 6 times higher kdis, while generally maintaining the binding affinities to C1q. TABLE 5: Binding affinities of different exemplary bispecific constructs Mutation KD(with respect to SEQ ID NO: kon(1 / Ms) kdis(1 / s) (M) SEQ ID NO: 10) N / A (non-humanized) SEQ ID NO: 2 2.38 4.85 105 1.16 10-3N / A SEQ ID NO: 10 20.7 3.22 105 6.67 10-3D31A, M33A SEQ ID NO: 14 25.8 3.09 105 7.96 10-3S32A, M33A SEQ ID NO: 15 17.4 8.44 105 1.47 10-2S32T, M33A SEQ ID NO: 16 34.1 3.77 105 1.28 10-2D31Q, M33A SEQ ID NO: 17 21.8 3.54 105 7.70 10-3D31N, M33A SEQ ID NO: 18 19.6 3.27 105 6.41 10-3M33A, Y109A SEQ ID NO: 28 105 2.81x1052.96x102M33A, N35A SEQ ID NO: 29 107 1.60x1051.72x102M33A, T28A SEQ ID NO: 30 122 2.69x1053.27x102M33A, R30A SEQ ID NO: 31 168 2.62x1054.40x102M33A, G112A SEQ ID NO: 32 178 3.17x1055.65x102M33A, W27A SEQ ID NO: 33 452 3.08x10513.3 M33A, R105A SEQ ID NO: 34 462 3.33x10515.4 M33A, F29A SEQ ID NO: 35 485 3.23x10515.7 M33A, S103A SEQ ID NO: 36 523 1.81x1059.48x102M33A, Y34A SEQ ID NO: 37 535 2.93x10515.7 M33A, T102A SEQ ID NO: 38 1490 2.31x10534.4 M33A, Y113A SEQ ID NO: 39 2960 1.74x10551.5 M33A, E114A SEQ ID NO: 40 NA NA NA M33A, Y115A SEQ ID NO: 41 NA NA NA M33A, D116A SEQ ID NO: 42 NA NA NA EXAMPLE 2: CYTOTOXICITY OF BISPECIFIC CONSTRUCTS TARGETING CD38 AND EGFR

[0303] Bispecific constructs were produced using the method provided in EXAMPLE 1 to investigate the ability of the constructs to facilitate complement-mediated cytotoxicity. TABLES 2-5 depicts exemplary bispecific constructs utilized in this example.

[0304] WSU-DLCL2 cells were maintained in high glucose Roswell Park Memorial Institute (RPMI) medium or RPMI 1640 + 10% heat inactivated FBS + 100U / ml Pen / Strep at 37°C, 8% CO2. Cells were maintained at 0.5-1.5 x 106 / ml and split 1:3-1:5 every 2-3 days. On the day of the CDC assay, cells were counted, centrifuged at 200 g for 5 minutes at room temperature (RT), washed with 30 ml cold RPMI, centrifuged for 5 minutes at 200×g at RT. Cells were then resuspended in cold RPMI + 9 mM MgCl2 at a concentration of 4 x 106 cells / ml. Cells were kept on ice before seeding at 25 microliters (uL) (0.1 x 105cells / well) into 96-well white OptiPlates (Revity). Each polypeptide was diluted in Veronal buffer (VB) + 9 mM MgCl2 and added to 13.3% (vol / vol) of normal human serum (NHS). The polypeptide constructs and serum (75 mL) were added to the cells to reach a final serum concentration of 10%, with construct dose typically ranging from 500-0.01 nM in 100 mL. As positive control for lysis, Digitonin (Promega, G9441) diluted to 0.13 mg / mL in CytoTox-Glo Assay buffer (Promega, G146) was further diluted to 0.4 mg / mL in VB + 9 mM MgCl2.75 mL of Digitonin was added to cells, giving a final concentrationof 0.03 mg / mL in 100 mL. As negative control for lysis, cells were incubated with 0 nM constructs in the respective serum conditions. The plates were incubated for 1 hour at 37°C, 8% CO2with a brief mixing for 5 seconds at 700 rpm after the first 30 min. Plates were equilibrated to RT for 10 minutes before wells were added 40 mL of AAF-Glo™ Substrate (Promega, CytoTox-Glo Kit, G9292). The plates were then briefly mixed for 5 seconds at 700 rpm, incubated at RT for 15-30 minutes, and luminescence was measured. MDA-MB-468 cells expressing EGFR were cultured in RPMI at 37° C with 8% CO2. All cells were supplemented with heat inactivated 10% fetal bovine serum and 0.1 mg / ml Penicillin-Streptomycin. Cells were detached for 10 min at 37° C. Cells were harvested, washed 2 times in PBS and resuspended in veronal buffer saline (VBS). For the CDC assay, 25000 cells were incubated with or without the bispecific construct and 15% NHS (final concentration) for 1 hour at 37° C. FIG.2B illustrates the effect of C1q binding affinity on WSU-DLCL2 cytotoxicity as a function of the concentration of the bispecific construct. As shown in FIG. 2B, the constructs with lower C1q binding affinity (i.e., binding affinities ranging from about 150 nM to about 1 μM) surprisingly displayed greater cytotoxicity at lower concentrations, relative to constructs with higher C1q binding affinity. FIG.5A - FIG.5B illustrates the effect of C1q binding affinity on WSU-DLCL2 cytotoxicity as a function of the concentration of the bispecific construct. As shown in FIG.5A, the constructs with lower C1q binding affinity results in better EC50 value on WSU-DLCL2 cells. Further, the low affinity bispecific constructs surprisingly displayed reduced bell-shaped curve. Moreover, as shown in FIG. 5B, high affinity bispecific constructs demonstrated more competition between C1q and CD38 bound bispecific construct. FIG. 6 illustrates the improvement in cytotoxic activity of bispecific constructs comprising low affinity C1q complement factor-engaging domains as compared to FDA-approved antibodies daratumumab and hexabody-38. As shown, BiCE spanning a range of affinities demonstrated higher max cytotoxicity and lysis than both HexaBody-CD38 and daratumumab on WSU-DLCL2 cells. Moreover, the increased cytotoxicity of the bispecific constructs was not limited to CD38. As shown in FIG. 9A, the bispecific construct comprising the complement factor-engaging domains with either low affinity to C1q binds to EGFR with fast on-and off-rates. Additionally, as observed in FIG. 9B introducing double mutations in the complement factor- engaging domain, as exemplified in PC463 and PC464, had a pronounced effect on the cytotoxic activity. The differences in antigen binding influences in vitro cytotoxic activity of bispecific constructs as disclosed herein (FIG. 10A - FIG. 10B). Bispecific constructs that bind both C1q and EGFR, were less affected by C1q affinity due to the high off-and on-rates of these constructs binding to EGFR, and demonstrated limited tendency to bell curve formation (FIG. 10A). Conversely, bispecific constructs that bind both C1q and CD38, were more affected by C1qaffinity due to the slow off-and on-rates of these constructs binding to CD38, and shows a bell curve when binding with high affinity to C1q (FIG.10B). Thus, the kinetics for the antigen binding moiety together with the kinetics for the C1q complement factor-engaging domain influences the cytotoxic activity of the bispecific constructs disclosed herein. EXAMPLE 3: CYTOTOXICITY OF BISPECIFIC CONSTRUCTS WITH MULTIPLE TARGETS

[0305] Bispecific constructs were produced and assessed using the methods provided in EXAMPLES 1 and 2. Bispecific constructs targeting CD38, CD20, CD19, CD7, FOLR1, EpCAM, HER2, and EGFR were assessed in specific cell lines (DND-41, Raji cells, IGROV1, SW403, OE19, A431). As demonstrated in FIG. 11A - FIG. 11H, the bispecific constructs were active with multiple targets, and all showed dose dependent cytotoxicity.

[0306] Thus, the foregoing examples demonstrates that bispecific constructs having a low affinity C1q complement factor-engaging domain antibody and a high affinity antigen binding moiety that binds a target antigen, such as a target protein (such as CD38) are more effective at inducing complement mediated cytotoxicity, relative to bispecific constructs having a high affinity C1q complement factor-engaging domain antibody and a high affinity antigen binding moiety that binds to the target antigen. Decreasing the affinity for C1q in the complement factor-engaging domain in the bispecific constructs as disclosed herein result in a less competitive inhibition at high concentrations of the constructs. Furthermore, decreasing the affinity for C1q as disclosed in the bispecific constructs have little impact on max cytotoxicity. Further, this example demonstrates that the bispecific constructs having low affinity C1q single domain antibodies and high affinity CD38 single domain antibodies are effective therapeutics for treating cancer characterized by overexpression of CD38. Additionally, the bispecific constructs as disclosed herein can further comprise albumin binding nanobodies that can bind to mouse, rat, cynomolgus monkey, or human albumin (as well as other species) to extend the serum half-life. EXAMPLE 4: CYTOTOXICITY OF CD19 TARGETING CONSTRUCTS

[0307] A CD-19 monoclonal antibody (CD19mAb) and a bispecific construct comprising the CD19mAb and two C1q-engaging domains (CD19-BiCE) as shown in FIG. 12 were produced and assessed using the methods provided in EXAMPLES 1 and 2.

[0308] Constructs targeting CD19 were assessed in Daudi cells according to the methods provided in EXAMPLE 3. As shown in FIG. 12, the CD19-BiCE enabled strong complement activation relative to the CD-19 monoclonal antibody alone.

[0309] Thus, the foregoing example demonstrates that bispecific constructs having a low affinity C1q complement factor-engaging domain antibody and a high affinity antigen binding moiety that binds CD19 are more effective at inducing complement mediated cytotoxicity, relative to CD19 binding antibodies. EXAMPLE 5: CYTOTOXICITY OF CD20 TARGETING CONSTRUCTS

[0310] A CD-20 monoclonal antibody (CD20mAb) and a bispecific construct comprising the CD20mAb and two C1q-engaging domains (CD20-BiCE) as shown in FIG.13 and FIG.14 were produced and assessed using the methods provided in EXAMPLES 1 and 2. The CD-20-BiCE is a fully human anti-CD20 (11B8 clone) fused to two humanized anti-C1q VHHs. The CD-20 BiCE can comprise an IgG1 or an IgG1 modified with a LALAPG to improve stability. The CD20-BiCE binds to human CD20 with an EC50 of 2 nM and has micromolar affinity for human C1q. The antibody is designed to deplete CD20 positive cells through CDC, CDCC, and CDCP of immune cells in a subject that is administered the CD20-BiCE. A CD20-BiCE with a LALA-PG mutation was also manufactured to mute all secondary effector functions of the IgG1 backbone.

[0311] Constructs targeting CD20 were assessed in primary B cells according to the methods provided in EXAMPLE 3. As shown in FIG. 13, the CD20-BiCE enabled strong complement activation relative to the CD-20 monoclonal antibody or rituximab alone.

[0312] In vitro activity of CD20-BiCE on human B cells and PBMCs was further evaluated (FIG. 15 and FIG. 16). CDC and B cell depletion was driven by VHH C1q engagement with some contribution from the C1q binding site in Fc portion of the bispecific CD20-BiCE. The CD20- BiCE also maintained intact antibody-dependent cellular cytotoxicity (ADCC) in human PBMCs (FIG. 17) showing that the CD-20 BiCE is also selective for the depletion of CD-20 expressing cells.

[0313] Human whole blood from 3 different donors was contacted with the CD20 mAb, CD-20- BiCE, or modified CD20- LALAPG BiCE at varying concentrations of 10 micrograms / milliliter (μg / ml), and 100 μg / ml. Alemtuzumab, a monoclonal antibody that targets CD52, was utilized as a positive control for B cell killing. Cetuximab, a monoclonal antibody that inhibits epidermal growth factor (EGFR) was utilized as a negative control. CD-20-BiCE increased the percentage of B cell killing by approximately 60% more than the CD20 mAb alone (FIG.18).

[0314] Thus, the foregoing example demonstrates that bispecific constructs having a low affinity C1q complement factor-engaging domain antibody and a high affinity antigen binding moiety that binds CD20 are more effective at inducing complement-mediated cytotoxicity, relative to CD20 monoclonal antibodies.EXAMPLE 6: EVALUATION OF BISPECIFIC C1Q-CD20 BiCE CONSTRUCTS IN NON-HUMAN PRIMATES IN VIVO.

[0315] Non-human cynomolgus primates were administered IV infusions at a dose of 25 milligrams / kilogram (mg / kg) of the CD20 IgG BiCE and the CD20 LALAPG IgG BiCE described in EXAMPLE 5. Each group of non-human primates (NHPs) were compared to a control group that did not received IV infusion of a bispecific construct. Blood was sampled periodically as shown in (FIG. 19). Lymph node biopsies were performed on day 15 and day 30. At the end of the evaluation, bone marrow and spleen tissues were removed from animals in each group.

[0316] Blood samples from NHPs were evaluated by flow cytometry for the presence of CD19 positive and CD20 positive cells over the course of 30 days (FIG. 20). Lymph node cells were also evaluated by flow cytometry for the presence of CD19 positive cells in all groups (FIG.21). All animals were successfully dosed and a rapid, sustained depletion of B cells was observed. Body weight remained the same for all animals with normal levels of hematological function and coagulation. No adverse effects were observed in NHPs that were treated with the bispecific constructs.

[0317] Further analysis of NHP cells in vitro showed that CDC was increased in CD20-BiCE and CD-20 BiCE LALAPG treated B cells (FIG. 22). Next, NHP B cells were evaluated following administration of different concentrations of CD-20 mAb, CD20-BiCE, and CD20-BiCE- LALAPG to non-naïve PBMCs with matched serum and Naiive PBMCs with matched serum. The percentage of B cells killed following administration to the cells was quantified for each concentration (FIG. 23A and FIG. 23B). Cells administered CD-20- BiCE and CD20-BiCE- LALAPG had a higher percentage of B cell killing relative to cells treated with CD20mAb or no treatment. The observed CDC and B cell depletion, was therefore driven by the C1q engagement of the CD-20 BiCE constructs.

[0318] Thus, the foregoing example demonstrates that bispecific constructs having a low affinity C1q complement factor-engaging domain antibody and a high affinity antigen binding moiety that binds CD20 are more effective at inducing complement-mediated cytotoxicity and B cell depletion in vivo in non-human primates, relative to NHPs that did not receive treatment. Moreover, the bispecific constructs also had a good safety profile in NHPs in vivo as characterized by the lack of adverse effects, change in body weight, or hematological function. EXAMPLE 7: CYTOTOXICITY OF CD38 TARGETING CONSTRUCTS

[0319] The following polypeptide constructs were manufactured in the table below and as shown in FIG.24 according to the methods in EXAMPLE 1. TABLE 6: CD-38 Constructs Name SEQ ID NOS: Targets Dara-BiCE C1q binding moiety: SEQ ID NO: 38 C1q Heavy and Light chain: SEQ ID CD-38 NOS: 71 and 72 HexaBody-CD38 CD-38 Daratumumab Heavy and Light chain: SEQ ID CD-38 NOS: 71 and 72 BiCE negative control C1q

[0320] Multiple tumor cell lines were obtain with varying levels of CD38 expression. Each cell line in Table 7 was tested in response to treatment with the polypeptide constructs in Table 6 and maximum cell lysis was quantified (FIG.25). CD38-IgG BiCE was superior to CD38-Hexabody and Daratumumab across a broad cell line panel for maximum cancer cell lysis. TABLE 7: Cell Lines in FIG.25. 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 leukemiaMono-mac-6 Acute 18383 4783 13005 monocytic leukemia OPM-2 Multiple 36224 10890 20522 myeloma LP-1 Multiple 191214 1997 26129 myeloma

[0321] A representative graph of complement dependent cytotoxicity observed in WSU-DLCL2 cells is shown in FIG.26. 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.27). Protein levels and max lysis of the tested cell lines were quantified (FIG.28).

[0322] Next, CD38 mAbs and CD38 BiCE versions listed in Table 8 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 8. CD38-targeting polypeptide and antibody constructs. Name Composition SEQ ID NOs: daratumumab Hc SEQ ID NO: 71 Dara Fab Lc SEQ ID NO: 117 10GS GS linker CM91-751 hNb78 (M33A,T102A) SEQ ID NO: 38 Felzartamab (MORO3087) Hc SEQ ID NO: 271 CM1253-1255 MORO3087 Lc SEQ ID NO: 272 Mezagitamab Hc SEQ ID NO: 273 CM1267-1269 Mezagitamab Lc SEQ ID NO: 274 CM313 Hc SEQ ID NO: 264 CM1781-1782 CM313 Lc SEQ ID NO: 265 CM313 Hc SEQ ID NO: 264 CM313 Lc SEQ ID NO: 265 5GS 5 X GS Linker CM1781-1783 hNb78 (M33A,T102A) SEQ ID NO: 38 SEQ ID NOS: 71 and SEQ ID Daratumumab Daratumumab NO: 72 CD38 Hc hexabody SEQ ID NO: 275 CM220-221 CD38 Lc SEQ ID NO: 276SEQ ID NO: 75 and SEQ ID Isatuximab Isatuximab NO: 76 Lc: light chain Hc: heavy chain GS: GS linker G: G linker See also Table B for sequences

[0323] 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.29). the CD38 BiCE showed high efficacy and potency, which is also better compared to CD38-Hexabody which display both lower max lysis and higher EC50value (See Table 9). TABLE 9. 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 B for sequences

[0324] Additional CD38 mAbs in Table 10 were evaluated in different IgG BiCE-formats as shown in FIG.30-FIG.31. TABLE 10. Exemplary CD38 monoclonal antibodies and polypeptide constructs. Name CompositionSEQ ID NO:CM1232-1234 Daratumumab (Dara 005) Hc and LcSEQ ID NO: 71 and SEQ ID NO: 72Nb75 (T53G)-10GS- Dara 005 Hc + Dara SEQ ID NO: 27; SEQ ID NO: 71; CM1233-1234 005 Lc and SEQ ID NO: 72 Dara (005) Hc + Dara modified Lc - 4G - SEQ ID NO: 71; SEQ ID NO: 109; CM1232-1235 hNb78(M33A,T102A) SEQ ID NO: 38Dara 005 Hc + Dara modified Lc - 5G - SEQ ID NO: 71; SEQ ID NO: 110; CM1232-1236 hNb78(M33A,T102A) SEQ ID NO: 38 Dara 005 Hc + Nb75(T53G)-10GS- Dara SEQ ID NO: 71; SEQ ID NO: 27; CM1232-1237 005 Lc SEQ ID NO: 72 CM1239-1241 Dara 003 Hc + Dara 003 LcSEQ ID NO: 102; SEQ ID NO: 103SEQ ID NO: 27; SEQ ID NO: 111; CM1240-1241 Nb75 (T53G)-10GS- 003 Hc + 003 Lc SEQ ID NO: 103 003 Hc + 003 Lc - 4G - SEQ ID NO: 102; SEQ ID NO: 103; CM1239-1242 hNb78(M33A,T102A) SEQ ID NO: 38 003 Hc + 003 Lc - 10GS - SEQ ID NO: 102; SEQ ID NO: 103; CM1239-1243 hNb78(M33A,T102A) SEQ ID NO: 38 SEQ ID NO: 102; SEQ ID NO: 27; CM1239-1244 003 Hc + Nb75(T53G)-10GS- 003 Lc SEQ ID NO: 103 CM1246-1248 MORO3080 Hc + MORO3080 LcSEQ ID NO: 104; SEQ ID NO: 105Nb75 (T53G)-10GS-MORO3080 Hc + SEQ ID NO: 27; SEQ ID NO: 104; CM1247-1248 MORO3080 Lc SEQ ID NO: 105 Lc: light chain Hc: heavy chain G: G linker See also Table B and Sequences section for sequences

[0325] As shown in FIG. 30 and FIG. 32, MORO3080 (e.g., CM1246-1248, SEQ ID NO: 104 and SEQ ID NO: 104) and daratumumab monoclonal antibodies (e.g., CM1232-1234, SEQ ID NO: 71 and SEQ ID NO: 72) do not work alone. However, adding Nb75 (T53G, SEQ ID NO: 27) 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, SEQ ID NO: 38) on the C terminus of the light chain of daratumumab potentiates CDC activity.

[0326] The IgG BiCEs shown in FIG. 32 exhibited high CDC activity on hard to kill CD38 positive cancer cell lines. EXAMPLE 8: DEPLETION OF CD38 BONE MARROW CELLS

[0327] Dara-BiCE was evaluated for killing of CD38 positive cells in a bone marrow from healthy human donors in the presence of complement (FIG.33). Dara-BiCE shows efficient complement mediated killing of plasmablasts and NK cells. Activity of Dara-BiCE and HexaBody-CD38 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 HexaBody-CD38 (FIG.34).EXAMPLE 9: ANTIBODY-DEPENDENT CELLULAR CYTOTOXICITY (ADCC) ACTIVITY OF CD38 TARGETING CONSTRUCTS

[0328] 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 11. TABLE 11. 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

[0329] 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.35A-FIG.35D. IgG BiCE constructs exhibited comparable ADCC activity relative to the parental mAbs (e.g., daratumumab (003) and MORO3080 as quantified in Table 12. TABLE 12: 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 10: EVALUATION OF CD38 TARGETING CONSTRUCTS ON CD38 ENZYMATIC ACTIVITY AND BINDING

[0330] 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.

[0331] 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 11: EPITOPE BINNING REVEALS COMBINATIONS FOR BIPARATOPIC CD38 IGG BICES.

[0332] 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 12: FLUID PHASE ACTIVATION AND SAFETY

[0333] BiCE polypeptide constructs were evaluated for complement activation in a fluid phase assay measuring C4a generation. The CD38-BiCEs exhibited specific complement activation inthe present of CD38-expressing cells (FIG.36). Thus, CD38 IgG BiCE constructs do not lead to complement activation in the absence of target cells.

[0334] 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 13: PHARMACOKINETIC EVALUATION OF DARA-BiCE IN A RAT MODEL.

[0335] The pharmacokinetic properties of Dara-BiCE was evaluated relative to daratumumab.

[0336] 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 13 below. TABLE 13. 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

[0337] 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 14: EVALUATION OF DARA-BICE TREATMENT OF CANCER IN AN SRG RAT MODEL

[0338] 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. 37. SRG rats have no T cells, B cells, or NK cells but have a fully active complement system.

[0339] 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 1stdose. The SRG rats treated with Dara-BiCE did not exhibit adverse reactions and maintained body weight.

[0340] While some embodiments of the bispecific constructs of present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodimentsare 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 bispecific constructs disclosed herein and that methods and structures within the scope of these claims and their equivalents be covered thereby.SPECIFIC EMBODIMENTS 1. An C1q complement factor-engaging polypeptide that comprises one or more modifications relative to a corresponding wild-type C1q complement factor-engaging polypeptide, said one or more modifications resulting in reduced binding affinity to a C1q complement factor as compared to a binding affinity of the corresponding wild-type C1q complement factor-engaging polypeptide to the C1q complement factor, wherein a cancer-antigen targeting bispecific construct comprising the C1q complement factor-engaging polypeptide exhibits an increased C1q-mediated immunological response to a cancer cell expressing a cancer antigen, as compared to a corresponding bispecific construct comprising the wild-type C1q complement factor-engaging polypeptide. 2. The C1q complement factor-engaging polypeptide of embodiment 1, wherein the increased C1q-mediated immunological response is determined by an in vitro assay that comprises: (a) linking the C1q complement factor-engaging polypeptide to an antigen binding moiety that binds the cancer antigen expressed by the cancer cell, thereby generating a bispecific construct that comprises the C1q complement factor-engaging polypeptide; (b) contacting the bispecific construct with the cancer cell in the presence of the C1q complement factor and determining an EC50 against the cancer cell; and (c) comparing the EC50of the bispecific construct that comprises the C1q complement factor- engaging polypeptide against the cancer cell to an EC50 of a control bispecific construct that comprises the corresponding wild-type C1q complement factor-engaging polypeptide linked to the antigen binding moiety that binds the cancer antigen. 3. The C1q complement factor-engaging polypeptide of embodiment 2, wherein the one or more modifications are present in a C1q binding region of the C1q complement factor-engaging polypeptide. 4. The C1q complement factor-engaging polypeptide of embodiment 1, wherein the C1q complement factor-engaging polypeptide has a binding affinity to the C1q complement factor of from about 10 nM to about 2 μM, as determined by biolayer interferometry. 5. The C1q complement factor-engaging polypeptide of any one of embodiments 1-4, wherein the C1q complement factor-engaging polypeptide comprises a chain constant region (CH), a heavy chain variable region (VH), a light chain constant region (CL), a light chain variable region(VL), a VHH domain, or a VNAR domain, a DARPin polypeptide, or a KNOB domain peptide. 6. The C1q complement factor-engaging polypeptide of any one of embodiments 1-5, wherein the one or more modifications comprises one or more amino acid substitutions relative to the corresponding wild-type C1q complement factor-engaging polypeptide. 7. The C1q complement factor-engaging polypeptide of embodiment 6, wherein the corresponding wild-type C1q complement factor-engaging polypeptide has an amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3. 8. The C1q complement factor-engaging polypeptide of embodiment 7, wherein the one or more amino acid substitutions are each at a position corresponding to a position in SEQ ID NO: 2 ranging from residue 32 to residue 114. 9. The C1q complement factor-engaging polypeptide of embodiment 7 or 8, wherein the one or more amino acid substitutions are each at a position corresponding to a position in SEQ ID NO: 2 selected from the group consisting of: residue 32, residue 53, residue 59, residue 74, residue 101, residue 102, residue 103, residue 104, residue 105, residue 106, residue 113, residue 114, and any combination thereof. 10. The C1q complement factor-engaging polypeptide of any one of embodiments 7-9, wherein the one or more amino acid substitutions are each at a position corresponding to a position in SEQ ID NO: 2 selected from the group consisting of: residue 101, residue 102, residue 113, residue 114, and any combination thereof. 11. The C1q complement factor-engaging polypeptide of any one of embodiments 7-10, wherein the one or more amino acid substitutions are selected from the group consisting of: D101A, T102A, Y113A, E114A, T102A / Y113A, T102A / E114A, and any combination thereof, as compared to the corresponding wild-type C1q complement factor-engaging polypeptide having the amino acid sequence of SEQ ID NO: 2. 12. The C1q complement factor-engaging polypeptide of any one of embodiments 7-11, wherein the one or more amino acid substitutions comprises the Y113A amino acid substitution, relative to the corresponding wild-type C1q complement factor-engaging polypeptide having theamino acid sequence of SEQ ID NO: 2. 13. The C1q complement factor-engaging polypeptide of any one of embodiments 7-12, wherein the C1q complement factor-engaging polypeptide comprises an amino acid sequence with about 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 4. 14. The C1q complement factor-engaging polypeptide of any one of embodiments 7-11, wherein the one or more amino acid substitutions comprises the E114A amino acid substitution, relative to the corresponding wild-type C1q complement factor-engaging polypeptide having the amino acid sequence of SEQ ID NO: 2. 15. The C1q complement factor-engaging polypeptide of any one of embodiments 7-12 or 14, wherein the C1q complement factor-engaging polypeptide comprises an amino acid sequence with about 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 5. 16. The C1q complement factor-engaging polypeptide of any one of embodiments 7-11, wherein the one or more amino acid substitutions comprises the D101A amino acid substitution, relative to the corresponding wild-type C1q complement factor-engaging polypeptide having the amino acid sequence of SEQ ID NO: 2. 17. The C1q complement factor-engaging polypeptide of any one of embodiments 7-12, 14 or 16, wherein the C1q complement factor-engaging polypeptide comprises an amino acid sequence with about 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 6. 18. The C1q complement factor-engaging polypeptide of an...

Claims

1. CLAIMS 1. A C1q complement factor-engaging polypeptide that comprises one or more modifications relative to a corresponding wild-type C1q complement factor-engaging polypeptide, said one or more modifications resulting in reduced binding affinity to a C1q complement factor as compared to a binding affinity of the corresponding wild-type C1q complement factor-engaging polypeptide to the C1q complement factor, wherein a cancer-antigen targeting bispecific construct comprising the C1q complement factor-engaging polypeptide exhibits an increased C1q-mediated immunological response to a cancer cell expressing a cancer antigen, as compared to a corresponding bispecific construct comprising the wild-type C1q complement factor-engaging polypeptide.

2. The C1q complement factor-engaging polypeptide of claim 1, wherein the increased C1q-mediated immunological response is determined by an in vitro assay that comprises: (a) linking the C1q complement factor-engaging polypeptide to an antigen binding moiety that binds the cancer antigen expressed by the cancer cell, thereby generating a bispecific construct that comprises the C1q complement factor-engaging polypeptide; (b) contacting the bispecific construct with the cancer cell in the presence of the C1q complement factor and determining an EC50 against the cancer cell; and (c) comparing the EC50of the bispecific construct that comprises the C1q complement factor-engaging polypeptide against the cancer cell to an EC50 of a control bispecific construct that comprises the corresponding wild-type C1q complement factor-engaging polypeptide linked to the antigen binding moiety that binds the cancer antigen.

3. The C1q complement factor-engaging polypeptide of claim 1, wherein the C1q complement factor-engaging polypeptide has a binding affinity to the C1q complement factor of from about 10 nM to about 2 μM, as determined by biolayer interferometry.

4. A bispecific construct comprising: (a) a C1q complement factor-engaging domain; and (b) an antigen-binding moiety that binds to a target antigen; wherein the C1q complement factor-engaging domain has a lower affinity for C1q complement factor, as compared to an affinity of the antigen-binding moiety to the target antigen, as measured by biolayer interferometry, and wherein the bispecific construct facilitates increased complement dependent cytotoxicity (CDC) via the C1q complement factor against a cell expressing the target antigen, as compared to an CDC level facilitated by otherwise comparable bispecific construct that comprises a C1q complement with a binding affinity for the C1q complement factor that is substantially equal to or greater than the binding affinity of the antigen- binding moiety to the target antigen.

5. The bispecific construct of claim 4, wherein: (a) the complement factor-engaging domain binds to C1q complement factor with a binding affinity of about 0.1 μM to about 2 μM, and the antigen-binding moiety binds to the target antigen with a binding affinity of less than 0.1 μM, as determined by biolayer interferometry; or (b) the bispecific construct binds to the C1q complement factor with a KDfrom 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.

6. The bispecific construct of claim 4, wherein the complement factor-engaging domain and the antigen-binding moiety are linked by a polypeptide linker sequence, optionally wherein the polypeptide linker sequence comprises: (a) at least 5 amino acids; (b) at least 30 amino acids; (c) about 10 amino acids to about 20 amino acids; or (d) an amino acid sequence of any one of SEQ ID NO: 46– SEQ ID NO:

62.

7. The bispecific construct of claim 4, further comprising an albumin binding nanobody (Nb), optionally wherein the albumin binding Nb is linked to the bispecific construct directly or indirectly at a N-terminal of the complement factor-engaging domain, between a C- terminal of the complement factor-engaging domain and a N-terminal of the antigen-binding moiety or a C-terminal of the antigen binding moiety.

8. The bispecific construct of claim 4, wherein the target antigen is a protein implicated in a disease, optionally wherein the disease is a cancer, an autoimmune disease, an inflammatory disease, or a neurological disease.

9. The bispecific construct of claim 4, wherein the target antigen is a protein expressed on the surface of a cell, optionally wherein the cell is: (a) a cancer cell, optionally wherein the cancer cell is a carcinoma cell, a breast cancer cell, an ovarian cancer cell, a gastric cancer cell, or a colon cancer cell; (b) an immune cell, optionally wherein the immune cell is a natural killer (NK) cell, a macrophage, a monocyte, a neutrophil, a dendritic cell, a T cell, a B cell, a mast cell, a basophil, or an eosinophil; (c) an endothelial cell; (d) an epithelial cell; or (e) a microbial cell.

10. The bispecific construct of claim 4, wherein the antigen-binding moiety binds to a cancer-specific marker, an immune-specific marker, a pathogen-specific marker, a tissue-specific marker, or an organ-specific marker; optionally wherein the antigen-binding moiety binds to EGFR, CD38, CD19, CD20, CD55, CD59, CD7, HER2, EpCAM, or FOLR1.

11. The C1q complement factor-engaging polypeptide of any one of claims 1-3 or the bispecific construct of any one of claims 4-10, wherein: (a) the C1q complement factor-engaging polypeptide or C1q complement factor-engaging domain modulates the activity of a complement system by directly or indirectly activating the complement system; (b) the C1q complement factor-engaging polypeptide or C1q complement factor-engaging domain directly binds a C1q complement factor; or (c) the C1q complement factor-engaging polypeptide or C1q complement factor-engaging domain indirectly binds a C1q complement factor.

12. The C1q complement factor-engaging polypeptide of any one of claims 1-3 or 11, or the bispecific construct of any one of claims 4-11, wherein: (a) the C1q complement factor-engaging polypeptide or C1q complement factor-engaging domain modulates the activity of a complement system, wherein the complement system is human complement system; and / or (b) the C1q complement factor is human C1q complement factor.

13. The C1q complement factor-engaging polypeptide of any one of claims 1-3 or 11- 12, or the bispecific construct of any one of claims 4-12, wherein the C1q complement factor- engaging polypeptide or C1q complement factor-engaging domain comprises a chain constant region (CH), a heavy chain variable region (VH), a light chain constant region (CL), a light chain variable region (VL), a VHH domain, or a VNARdomain, a DARPin polypeptide, or a KNOB domain peptide.

14. The C1q complement factor-engaging polypeptide of any one of claims 1-3 or 11- 13, or the bispecific construct of any one of claims 4-13, wherein: (a) one or more modifications are present in a C1q binding region of the C1q complement factor-engaging polypeptide or C1q complement factor-engaging domain relative to the sequence of the corresponding wild-type C1q complement factor-engaging polypeptide; and / or (b) the C1q complement factor-engaging polypeptide or C1q complement factor-engaging domain comprises one or more modifications comprising one or more amino acid substitutions relative to the sequence of the corresponding wild-type C1q complement factor- engaging polypeptide.

15. The C1q complement factor-engaging polypeptide of any one of claims 1-3 or 11- 14, or the bispecific construct of claim 14, wherein the corresponding wild-type C1q complement factor-engaging polypeptide has an amino acid sequence of SEQ ID NO: 2 or SEQ ID NO:

3.

16. The C1q complement factor-engaging polypeptide of any one of claims 1-3 or 11- 15 or the bispecific construct of claim 14 or 15, wherein: (a) the one or more amino acid substitutions are each at a position corresponding to a position in SEQ ID NO: 2 ranging from residue 32 to residue 114; (b) the one or more amino acid substitutions are each at a position corresponding to a position in SEQ ID NO: 2 selected from the group consisting of: residue 32, residue 53, residue 59, residue 74, residue 101, residue 102, residue 103, residue 104, residue 105, residue 106, residue 113, residue 114, and any combination thereof; (c) the C1q complement factor-engaging polypeptide or C1q complement factor-engaging domain comprises one or more amino acid substitutions selected from amino acid substitutions at positions corresponding to residue 32, residue 53, residue 59, residue 74, residue 101, residue 102, residue 103, residue 104, residue 105, residue 106, residue 113, and residue 114, of SEQ ID NO: 2; (d) the one or more amino acid substitutions are each at a position corresponding to a position in SEQ ID NO: 2 selected from the group consisting of: residue 101, residue 102, residue 113, residue 114, and any combination thereof; (e) the C1q complement factor-engaging polypeptide or C1q complement factor-engaging domain comprises one or more amino acid substitutions selected from amino acid substitutions at positions corresponding to residue 101, residue 102, residue 113, residue 114, in SEQ ID NO: 2; (f) the one or more amino acid substitutions are selected from the group consisting of: D101A, T102A, Y113A, E114A, T102A / Y113A, T102A / E114A, and any combination thereof, as compared to the corresponding wild-type C1q complement factor- engaging polypeptide having the amino acid sequence of SEQ ID NO: 2; or (g) the C1q complement factor-engaging polypeptide or C1q complement factor-engaging domain comprises one or more amino acid substitutions selected from the group consisting of: D101A, T102A, Y113A, E114A, T102A / Y113A, T102A / E114A, , as compared to the corresponding wild-type C1q complement factor-engaging polypeptide having the amino acid sequence of SEQ ID NO:

2.

17. The C1q complement factor-engaging polypeptide of any one of claims 1-3 or 11- 16, or the bispecific construct of any one of claims 14-16, wherein: (a) the one or more amino acid substitutions comprises the Y113A amino acid substitution, relative to the corresponding wild-type C1q complement factor-engaging polypeptide having the amino acid sequence of SEQ ID NO: 2, optionally wherein the C1q complement factor engaging polypeptide or C1q complement factor-engaging domain comprises an amino acid sequence with about 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 4; (b) the one or more amino acid substitutions comprises the E114A amino acid substitution, relative to the corresponding wild-type C1q complement factor-engaging polypeptide having the amino acid sequence of SEQ ID NO: 2, optionally wherein the C1q complement factor engaging polypeptide or C1q complement factor-engaging domain comprises an amino acid sequence with about 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 5; (c) the one or more amino acid substitutions comprises the D101A amino acid substitution, relative to the corresponding wild-type C1q complement factor-engaging polypeptide having the amino acid sequence of SEQ ID NO: 2, optionally wherein the C1q complement factor engaging polypeptide or C1q complement factor-engaging domain comprises an amino acid sequence with about 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 6; (d) the one or more amino acid substitutions comprises the T102A / Y113A amino acid substitutions, relative to the corresponding wild-type C1q complement factor-engaging polypeptide having the amino acid sequence of SEQ ID NO: 2, optionally wherein the C1q complement factor engaging polypeptide or C1q complement factor-engaging domain comprises an amino acid sequence with about 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO: 7; or (e) the one or more amino acid substitutions comprises the T102A / E114A amino acid substitutions, relative to the corresponding wild-type C1q complement factor-engaging polypeptide having the amino acid sequence of SEQ ID NO: 2, optionally wherein the C1q complement factor engaging polypeptide or C1q complement factor-engaging domain comprises an amino acid sequence with about 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO:

8.

18. The C1q complement factor-engaging polypeptide of any one of claims 1-3 or 11- 17, or the bispecific construct of any one of claims 4-17, wherein the C1q complement factor- engaging polypeptide or C1q complement factor-engaging domain comprises: (a) an amino acid sequence of any one of SEQ ID NO: 4 to SEQ ID NO: 42; (b) an amino acid sequence having at least about 90%, 95%, 97%, 99% or 100% sequence identity to any one of SEQ ID NO: 4 to SEQ ID NO: 42; or (c) an amino acid sequence having at least about 90%, 95%, 97%, 99% or 100% sequence identity to any one of SEQ ID NO: 1 to SEQ ID NO:

3.

19. An engineered C1q complement factor-engaging polypeptide comprising: (a) one or more amino acid substitutions relative to a corresponding wildtype C1q complement factor-engaging polypeptide comprising SEQ ID NO: 2, wherein the one or more amino acid substitutions are at a position corresponding to a position in SEQ ID NO: 2 ranging from residue 32 to residue 114; and (b) a binding affinity to the C1q complement factor of from about 150 nM to about 1 μM, as compared to a binding affinity of the corresponding wildtype C1q complement factor-engaging polypeptide to the C1q complement factor, as determined by biolayer interferometry, wherein a cancer-antigen targeting bispecific construct comprising the engineered C1q complement factor-engaging polypeptide exhibits an increased C1q-mediated immunological response to a cancer cell expressing a cancer antigen, as compared to a corresponding bispecific construct comprising the wildtype C1q complement factor- engaging polypeptide.

20. A composition comprising: (a) the engineered C1q complement factor-engaging polypeptide of claim 19; and (b) an antigen-binding moiety linked to the C1q complement factor-engaging polypeptide, wherein the antigen-binding moiety binds to CD-19.

21. A composition comprising: (a) the engineered C1q complement factor-engaging polypeptide of claim 19; and (b) an antigen-binding moiety linked to the C1q complement factor-engaging polypeptide, wherein the antigen-binding moiety binds to CD-20.

22. A composition comprising: (a) the engineered C1q complement factor-engaging polypeptide of claim 19; and (b) an antigen-binding moiety linked to the C1q complement factor-engaging polypeptide, wherein the antigen-binding moiety binds to CD-38.

23. A pharmaceutical composition that comprises: the bispecific construct of any one of claims 4-19; the engineered C1q complement factor-engaging polypeptide of claim 19; and / or the composition of any one of claims 20-22; and optionally, a pharmaceutically acceptable diluent, carrier, or excipient.

24. A composition that comprises: (I) a bispecific construct of any one of claims 4-18; or (II) a bispecific construct that comprises: (a) a complement factor-engaging domain that binds to a C1q complement factor with a binding affinity of about 0.1 μM, as determined by biolayer interferometry; and (b) an antigen-binding moiety linked to the complement factor- engaging domain, wherein the antigen-binding moiety binds to a target antigen expressed by a cell with a binding affinity of less than 0.1 μM, as determined by biolayer interferometry; optionally wherein the target antigen is a cancer-specific marker, an immune- specific marker, an organ-specific marker, or a protein implicated in a disease, and optionally wherein the cancer specific marker is selected from the group consisting of: CD7, CD19, CD20, CD38, EGFR, EpCAM, FOLR1 and HER2; optionally wherein the composition further comprises an albumin-binding moiety.

25. A method of enhancing anti-cancer effect or the anti-inflammatory effect of an antibody, the method comprising fusing the antibody to a complement factor-engaging domain, wherein: (a) the complement factor engaging domain is a C1q complement factor- engaging polypeptide according to any one of claims 1-3 or 11-19; or (b) the complement factor-engaging domain has a lower affinity for a complement factor as compared to an affinity of the antibody for its target antigen, optionally wherein the antibody or fragment thereof has a sequence of any one of SEQ ID NO: 63 – SEQ ID NO:

99.

26. A composition for use in inducing complement-mediated cancer cell death of a cancer cell that expresses a cancer-specific marker, the composition comprising: (I) a bispecific construct according to any one of claims 4-18, wherein the target antigen is a cancer-specific marker expressed by the cancer cell; or (II) a bispecific construct that comprises: (i) a complement factor-engaging domain that binds to a human C1q complement factor with a binding affinity of about 1 μM, and (ii) an antigen binding moiety that binds to the cancer-specific marker expressed by the cancer cell; wherein contacting of the bispecific construct with the cancer cell results in complement-mediated cell death of the cancer cell that expresses the cancer-specific marker.

27. A composition for use in treating an autoimmune disease in a subject in need thereof, the composition comprising: (I) a bispecific construct according to any one of claims 4-18, wherein the target antigen is an autoimmune marker expressed by a cell; or (II) a bispecific construct that comprises: (a) a complement factor-engaging domain that binds to human C1q complement factor with a binding affinity of about 1 μM, as determined by biolayer interferometry (BLI); and (b) an antigen-binding moiety that binds to an autoimmune marker expressed by a cell; wherein the bispecific construct treats the autoimmune disease when administered to the subject, optionally wherein the autoimmune disease is multiple sclerosis, psoriasis, psoriatic arthritis, rheumatoid arthritis, systemic lupus erythematosus, Crohn's disease, Sjogren's syndrome, Behcet's disease, ulcerative colitis, or Guillain-Barre syndrome.

28. A composition for use in treating cancer characterized by aberrant expression of a cancer-specific marker by a cancer cell in a subject in need thereof, the composition comprising: (I) a bispecific construct according to any one of claims 4-18, wherein the target antigen is a cancer-specific marker expressed by the cancer cell; or (II) a bispecific construct that comprises: (a) a complement factor-engaging domain that binds to human C1q complement factor with a binding affinity of about 1 μM, as determined by biolayer interferometry; and (b) an antigen-binding moiety that binds to the cancer specific marker expressed by the cancer cell; wherein the bispecific construct treats the cancer when administered to the subject; and optionally wherein the cancer-specific marker is: (i) CD19; (ii) CD20; (iii) CD38; (iv) EGFR; or (v) selected from the group consisting of CD7, CD19, CD20, CD38, CD55, CD59, EGFR, EpCAM, FOLR1, and HER2.

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