CFD-c2 binding molecules

Abstract

Provided herein are dual-antigen binding molecules, and nucleic acid sequences encoding such molecules, that bind human complement factor D (FD) and human complement component 2 (C2). In particular embodiments, the FD binding region of the dual-antigen binding molecules comprises light and heavy chain variable regions, and the C2 binding region comprises a first single monomeric variable antibody domain (aka VHH), and optionally second single monomeric variable antibody domain. In certain embodiments, the dual-antigen binding molecules are used to treat complement-related diseases (e.g., dysregulated complement activation diseases).

Description

[0001] Attorney Docket Number: CCF-43777.601

[0002] CFD-C2 BINDING MOLECULES

[0003] CROSS REFERENCE TO RELATED APPLICATION

[0004] The present application claims priority to U.S. provisional application serial number 63 / 736,386 filed December 19, 2024, which is herein incorporated by reference in its entirety.

[0005] SEQUENCE LISTING PARAGRAPH

[0006] The text of the computer readable sequence listing filed herewith, titled “CCF_43777_601_SequenceListing.xml”. created December 16, 2025, having a file size of 12,660 bytes, is hereby incorporated by reference in its entirety.

[0007] FIELD OF THE INVENTION

[0008] Provided herein arc dual-antigen binding molecules (c.g., antibody-nanobody binding molecules), and nucleic acid sequences encoding such molecules, that bind human complement factor D (FD) and human complement component 2 (C2). In particular embodiments, the FD binding region of the dual-antigen binding molecules comprises light and heavy chain variable regions, and the C2 binding region comprises a first single monomeric variable antibody domain (aka VHH), and optionally second single monomeric variable antibody domain. In certain embodiments, the dual-antigen binding molecules are used to treat complement-related diseases (e.g., dysregulated complement activation diseases).

[0009] BACKGROUND OF THE INVENTION

[0010] Complement, a key component of the innate immune system, acts as the first line of defense against infections. Complement components are primarily produced by hepatocytes and secreted into the blood as zymogens, thus need to be activated to be functional. There are three complement activation pathways, i.e., the classical, lectin and alternative pathways, which are initiated by different mechanisms, but all converge to generate the same set of effector molecules. First, activated complement generates large numbers of activated C3 proteins (C3b / iC3b) that bind covalently to the target cells, opsonizing them to promote engulfment by phagocytes that express C3 receptors. Second, activation of C3 and the downstream activation of C5 release anaphylatoxins such as C3a and C5a, to recruit and activate myeloid cells to the site of complement activation to promote inflammation and Attorney Docket Number: CCF-43777.601 phagocytosis through their own set of receptors. Third, activated C3 leads to the activation of C5 and the assembly of C5b-9, a.k.a., membrane attack complexes (MACs), to directly damage target cells by forming "pores" on the cell surfaces13. Apparently, C3 is the central component of all three complement activation pathways and required for complement activation.

[0011] Like other parts of the immune system, excessive complement activation causes diseases. C3 thus has emerged as an attractive therapeutic target in complement-mediated diseases41, because inhibiting C3 completely blocks all complement activation pathways and suppresses all C3b / iC3b-mediated phagocytosis, C3a / C5a-associated inflammation and MAC-induced cellular damage to yield a robust therapeutic effect. In contrast, targeting C5 only reduces C5a production and MAC formation, while targeting Cis only inhibits the classical pathway. Indeed, a peptide-based C3 inhibitor (pegcetacoplan) was recently approved for treating PNH, and it showed good safety and great efficacy in inhibiting both cxtravascular hemolysis (mediated by C3b / iC3b) and intravascular hemolysis (mediated by the MAC) in PNH patients42-44, with the result that only 15% of the patients still require blood transfusions. In addition, whereas the anti-C5 mAbs did not effectively treat geographic atrophy45, pegcetacoplan was shown to achieve a mild but statistically significant slowing of GA in patients46,47, and was recently approved to be the first drug effective against GA. However, targeting C3 presents significant challenges. In the blood, C3 is the most abundant complement protein, present at concentrations of 1000-1500 ug / inL Consequently, a large dosage of this C3 inhibitor (>2 grams per week) is needed to treat diseases such as PNH48, and severe adverse effects (retinal vasculitis) in GA patients were reported. Therefore, it would be substantially better to achieve the same C3 -inhibiting effects, namely inhibiting all C3b / iC3b-mediated opsonization, C3a / C5a-promoted inflammation, and MAC- mediated cell damage, without targeting C3.

[0012] SUMMARY OF THE INVENTION

[0013] Provided herein are dual-antigen binding molecules, and nucleic acid sequences encoding such molecules, that bind human complement factor D (FD) and human complement component 2 (C2). In particular embodiments, the FD binding region of the dual-antigen binding molecules comprises light and heavy chain variable regions, and the C2 binding region comprises a first single monomeric variable antibody domain (aka VHH), and optionally second single monomeric variable antibody domain. In certain embodiments, the Attorney Docket Number: CCF-43777.601 dual-antigen binding molecules are used to treat complement-related diseases (e.g., dysregulated complement activation diseases).

[0014] In some embodiments, provided herein are compositions comprising: a dual-antigen binding molecule, or one or more nucleic acid molecules encoding the dual-antigen binding molecule, wherein the dual-antigen binding molecule comprises: i) a human complement factor D (FD) binding region (e.g., at least antigen binding region from antibody Lampalizumab; see US Pat. 8,273,352, herein incorporated by reference) comprising: A) an antibody light chain comprising a light chain variable region, B) an antibody heavy chain comprising a heavy chain variable region and at least one heavy chain constant region; ii) a human complement component 2 (C2) binding region comprising: A) a first single monomeric variable antibody domain (SMVAD1) (e.g., any one shown in WO2024186861, herein incorporated by reference, including SMVADs, or CDRs therefrom, from Figures 8 and 10-14, which are specifically incorporated by reference herein), and B) optionally a second single monomeric variable antibody domain (SMVAD2) which, if present, is joined to the SMVAD1 by a second linker; and iii) a first linker attached to the at least one heavy constant region and to the SMVAD1. In some embodiments, the SMVAD1 and SMVAD2 are identical or nearly identical. In particular embodiments, the human complement factor D (FD) binding region comprises at least antigen binding region from an antibody described in any of: US Pat. 8,273,352; US Pat. 10,407,510; US Pat. Pub. 20200115462; US Pat. Pub. US20170143843; U.S. 10,179,821; and U.S. Pat. Pub. 2018 / 0334496, all of which are herein incorporated by reference, particularly for complement factor D antibody binding regions.

[0015] In certain embodiments, the light chain variable region comprises at least one of the following: i) a CDR1 amino acid sequence comprising SEQ ID NO:9 (STDIDDD), or SEQ ID NO:9 with one conservative amino acid change; ii) a CDR2 amino acid sequence comprising SEQ ID NO: 10 (GGN), or SEQ ID NO: 10 with one conservative amino acid change; iii) a CDR3 amino acid sequence comprising SEQ ID NO: 11 (LQSDSLPYT), or SEQ ID NO:11 with one conservative amino acid change. In additional embodiments, the light chain variable region comprises SEQ ID NO:8, or SEQ ID NO:8 with one, two, or three conservative amino acid changes. In particular embodiments, the light chain variable region comprises four Framework regions, wherein, for example, the four Framework regions are humanized, or human Framework regions. In other embodiments, the antibody light chain comprises a light chain constant region.

[0016] In further embodiments, the heavy chain variable regions comprises at least one of (or two of, or all three of) the following: i) a CDR1 amino acid sequence comprising SEQ ID Attorney Docket Number: CCF-43777.601

[0017] N0:2 (GYTFTNYG), or SEQ ID N0:2 with one conservative amino acid change; ii) a CDR2 amino acid sequence comprising SEQ ID NO:3 (INTYTGET), or SEQ ID NO:3 with one conservative amino acid change; and iii) a CDR3 amino acid sequence comprising SEQ ID NO:4 (CEREGGVNN), or SEQ ID NO:4 with one conservative amino acid change. In further embodiments, the heavy chain variable region comprises four Framework regions, wherein the four Framework regions are humanized, or human Framework regions.

[0018] In certain embodiments, the SMVAD1 and / or SMVAD 2 comprises at least one of the following: i) a CDR1 amino acid sequence comprising SEQ ID NO:5 (GHTFRSY), or SEQ ID NO:5 with one conservative amino acid change; ii) a CDR2 amino acid sequence comprising SEQ ID NO:6 (SWTGGS), or SEQ ID NO:6 with one conservative amino acid change; iii) a CDR3 amino acid sequence comprising SEQ ID NO:7 (DYSGDFLLSDLDS), or SEQ ID NO:7 with one conservative amino acid change. In particular embodiments, the SMVAD 1 and SMVAD2 further comprise four Framework regions, wherein the four Framework regions arc camclid, humanized, or human Framework regions. In other embodiments, the SMVAD1 and / or SMVAD 2 comprises SEQ ID NO:12, or SEQ ID NO:12 with one conservative amino acid change.

[0019] In particular embodiments, the at least one heavy chain constant region comprises three heavy chain constant regions (e.g., a CHI, a CH2, and a CH3). In other embodiments, the composition comprises two of the dual-antigen binding molecules (e.g., such that the at least one heavy chain constant region on each are aligned with each other). In further embodiments, the compositions further comprise a physiologically tolerable buffer. In additional embodiments, the composition comprises the one or more nucleic acid molecules, and optionally the composition further comprises an expression vector, and wherein the first and / or second nucleic acid sequences are present in the expression vector. In particular embodiments, the composition comprises the dual-antigen binding molecule.

[0020] In some embodiments, provided herein are methods of treating or preventing a complement-related disease or condition comprising: treating a subject with a composition comprising the dual-antigen binding molecule, or an expression vector comprising the one or more nucleic acid molecules encoding the dual-antigen binding molecule, as described above or anywhere herein, and wherein the subject (e.g., human subject) has, or is suspected to develop, a complement-related disease or condition.

[0021] In other embodiments, the complement-related disease comprises a dysregulated complement activation disease. In additional embodiments, the complement-related disease comprises myasthenia gravis or atypical hemolytic uremic syndrome. In some embodiments, Attorney Docket Number: CCF-43777.601 the complement-related disease comprises paroxysmal nocturnal hemoglobinuria (PNH) or and autoimmune hemolytic anemia (AIHA). In further embodiments, the composition comprises the expression vector, and wherein the first and / or second nucleic acid sequences are present in the expression vector. In other embodiments, the composition comprises the dual-antigen binding molecule.

[0022] DESCRIPTION OF THE FIGURES

[0023] Figure 1A shows a diagram of an exemplary C2 / CFD bispecific nanobody-mAb construct, which is an anti-anti-Complement Factor D antibody (e.g., Lampalizumab) with two anti -complement component 2 (C2) VHH nanobody sequences attached to each of the C3 heavy chains. Figure IB shows the amino acid sequence of an exemplary heavy chain of the anti-CFD antibody (Lampalizumab in this case) attached at the C3 heavy chain to two anti-C2 VHH sequences (SEQ ID NO:1). Figure 1C shows the amino acid sequence of an exemplary light chain of the anti-CFD antibody ((Lampalizumab in this case) (SEQ ID NO:8).

[0024] Figure 2: The exemplary C2 / CFD bispecific nanobody-mAb herein was compared with the parental anti-C2 nAb IB 10, parental Lampalizumab, and AMY- 101, a clinical stage C3 inhibitor in both CP (Fig. 2A) and AP-mediated hemolysis assays (Fig. 2B).

[0025] Figure 3: The exemplary C2 / CFD bispecific nanobody-mAb was compared with the parental anti-C2 nAb IB 10, parental Lampalizumab, and AMY- 101, a clinical stage C3 inhibitor in both CP (Fig. 3 A) and AP-mediated hemolysis assays (Fig. 3B).

[0026] Figure 4: The exemplary C2 / CFD bispecific nAb-mAb was compared with AMY- 101 (a clinical stage C3 inhibitor), Ravlizumab (an approved C5 inhibitor), and LNP23 (an approved CFB inhibitor) in both CP (Fig. 4A) and AP-mediated (Fig. 4B) hemolysis assays.

[0027] Figure 5: The exemplary C2 / CFD bispecific nAb-mAb herein was compared with AMY-101 (a clinical stage C3 inhibitor), Ravlizumab (an approved C5 inhibitor), and LNP23 (an approved CFB inhibitor) in both CP (Fig. 5A) and AP-mediated (Fig. 5B) hemolysis assays.

[0028] Figure 6: The exemplary C2 / CFD bispecific nAb-mAb was compared with AMY- 101 (a clinical stage C3 inhibitor) and LNP23 (an approved CFB inhibitor) in inhibiting complement activation (C3b deposition) on endothelial cells.

[0029] Figure 7: The exemplary C2 / CFD bispecific nAb-mAb was compared with AMY- 101 (a clinical stage C3 inhibitor) and LNP23 (an approved CFB inhibitor) in inhibiting complement activation-mediated endothelial cell damages. Attorney Docket Number: CCF-43777.601

[0030] Figure 8: shows a comparison of the exemplary C2-CFD bispecific Ab with AMY- 101, a clinical stage C3 inhibitor in an in vitro CP-mediated hemolysis assay, showing that while AMY-101 did not have any observed inhibitor activity at the highest concentration tested, the bispecific C2-CFD Ab potently inhibited CP complement-mediated hemolysis

[0031] Figure 9: shows a comparison of the exemplary C2-CFD bispecific Ab with AMY- 101, a clinical stage C3 inhibitor in an in vivo CP-mediated hemolysis model, showing that at the dose given, the bispecific Ab potently inhibited CP complement-mediated hemolysis in vivo. Levels of released blood hemoglobin measured by OD414 in sera samples from mice treated with 1.2uM C3 inhibitor (AMY-101) or the bispecific C2-CFD Ab.

[0032] Figure 10: The C2 / CFD bispecific nAb-mAb was compared with AMY-101, a clinical stage C3 inhibitor in inhibiting complement mediated hemolysis in vivo.

[0033] DEFINITIONS

[0034] To facilitate an understanding of the invention, a number of terms arc defined below.

[0035] The term "antibody," as used herein, is distinct from “nanobody” and “single-domain antibody,” and is intended to refer to immunoglobulin molecules comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CHI, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VII and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). Each variable region (VH or VL) contains 3 CDRs, designated CDR1, CDR2 and CDR3 (see, Figures IB and 1C). Each variable region also contains 4 framework sub-regions, designated FR1, FR2, FR3 and FR4 (see, Figures IB and 1C), which may be human framework sub-regions.

[0036] As used herein, the term "antibody fragment or portion or antigen binding region" refers to a portion of an intact antibody. Examples of antibody fragments or portions include, but are not limited to, linear antibodies, single-chain antibody molecules, Fv, Fab and F(ab')2 fragments, and multi-specific antibodies formed from antibody fragments. The antibody fragments preferably retain at least part of the heavy and / or light chain variable region. Attorney Docket Number: CCF-43777.601

[0037] A “nanobody,” or “single variable domain” (“VHH”) or “single monomeric variable antibody domain” (“SMVAD”) or “single domain antibody” as used herein, refer to the smallest antigen binding fragment originally derived from a naturally occurring heavy chain antibody and is known to the person skilled in the art. Such nanobodies can be derived from antibodies raised in Camelidae species, for example in camel, llama, dromedary, alpaca and guanaco. Nanobodies may also be synthetically produced, such as by overexpression in bacteria. Single domain antibodies are antibodies whose complementary determining regions (CDRs) are part of a single domain polypeptide. Examples include, but are not limited to, heavy chain antibodies, antibodies naturally devoid of light chains, single domain antibodies derived from conventional 4-chain antibodies, engineered antibodies and single domain scaffolds other than those derived from antibodies.

[0038] The phrase "nanobody- monoclonal antibody," or "nAb-mAb," as used herein refers to a construct that has at least part of an antibody binding region (e.g., Fv or F'ab) and at least part of a nanobody (e.g., VHH). In certain embodiments, the "nAb-mAb," (or mAb-nAb) comprises at least a Fv region of antibody linked to at least one VHH, or two VHHs. In certain embodiments, nAb-Mabs bind to both complement Factor D (CFD) or (FD) and bind to complement component 2 (C2).

[0039] As used herein, the terms "complementarity determining region" and "CDR" refer to the regions that are primarily responsible for antigen-binding. There are three CDRs in a light chain variable region (CDRE1, CDRE2, and CDRE3), and three CDRs in a heavy chain variable region (CDRH1, CDRH2, and CDRH3). In single monomeric variable antibody domains, there are three CDRs (CDR1, CDR2, and CDR3).

[0040] As used herein, the term "framework" refers to the residues of the variable region other than the CDR residues. There are four separate framework sub-regions that make up the framework: FR1, FR2, FR3, and FR4 (see, e.g., Figures IB and 1C). In antibodies or fragments thereof, as opposed to nanobodies, in order to indicate if the framework sub-region is in the light or heavy chain variable region, an "E" or "H" may be added to the sub-region abbreviation (e.g., "FREI" indicates framework sub-region 1 of the light chain variable region). It is noted that, in certain embodiments, the FD binding regions of the present invention may have less than a complete framework (e.g. may have a portion of a framework that only contains one or more of the four sub-regions).

[0041] As used herein, the term "fully human framework" means a framework with an amino acid sequence found naturally in humans. Examples of fully human frameworks, include, but are not limited to, KOL, NEWM, REI, EU, TUR, TEI, LAY and POM (See, e.g., Kabat et al., Attorney Docket Number: CCF-43777.601

[0042] (19 1) Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA; and Wu et al., (1970) J. Exp. Med. 132, 211-250, both of which are herein incorporated by reference). In certain embodiments, the dual-antigen binding molecules herein (e.g., mAb-nAb) binding molecules herein have fully human framework.

[0043] As used herein, the terms "subject" and "patient" refer to any animal, such as a mammal like a dog, cat, bird, livestock, and preferably a human.

[0044] As used herein, the term "codon" or "triplet" refers to a group of three adjacent nucleotides which specify one of the naturally occurring amino acids found in polypeptides. The term also includes codons which do not specify any amino acid. It is also noted that, due to the degeneracy of the genetic code, there are many codons that code for the same amino acid (see, e.g., amino acid sequences in Figures IB and 1C). As such, many of the bases of the nucleic acid sequences of the present invention can be changed without changing the actual amino acid sequence that is encoded. The present disclosure is intended to encompass all such nucleic acid sequences.

[0045] As used herein, the temrs "an oligonucleotide having a nucleotide sequence encoding a polypeptide," "polynucleotide having a nucleotide sequence encoding a polypeptide," and "nucleic acid sequence encoding a peptide" means a nucleic acid sequence comprising the coding region of a particular polypeptide. The coding region may be, for example, present in a cDNA, genomic DNA, or RNA form. When present in a DNA form, the oligonucleotide or polynucleotide may be single-stranded (i.e., the sense strand) or double- stranded. Suitable control elements such as enhancers / promoters, splice junctions, polyadenylation signals, etc. may be placed in close proximity to the coding region of the gene if needed to permit proper initiation of transcription and / or correct processing of the primary RNA transcript. Alternatively, the coding region utilized in the expression vectors of the present invention may contain endogenous enhancers / promoters, splice junctions, intervening sequences, polyadenylation signals, etc., or a combination of both endogenous and exogenous control elements.

[0046] DESCRIPTION OF THE INVENTION

[0047] Provided herein are dual-antigen binding molecules, and nucleic acid sequences encoding such molecules, that bind human complement factor D (FD) and human complement component 2 (C2). In particular embodiments, the FD binding region of the dual-antigen binding molecules comprises light and heavy chain variable regions, and the C2 binding region comprises a first single monomeric variable antibody domain (aka VHH), and Attorney Docket Number: CCF-43777.601 optionally second single monomeric variable antibody domain. In certain embodiments, the dual-antigen binding molecules are used to treat complement-related diseases (e.g., dysregulated complement activation diseases).

[0048] In certain embodiments, the dual-antigen binding molecules herein comprise one or more of the antibodies, variable regions, or CDRs shown in SEQ ID NOS: 1-12 and / or variable regions or CDRs with one or more conservative or non-conservative amino acid changes in these SEQ ID NOS: 1-12, and nucleic acid sequences encoding SEQ ID NOs: l-12. Changes to the amino acid sequences of the CDRs or variable regions may be generated by changing the nucleic acid sequence encoding the amino acid sequence. A nucleic acid sequence encoding a variant of a given CDR or variable region may be prepared by methods known in the art using the guidance of the present specification for particular sequences. These methods include, but are not limited to, preparation by site-directed (or oligonucleotide-mediated) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared nucleic acid encoding the CDR or variable region.

[0049] The nanobodies (SMVADs) portion of the binding molecules herein, according to the present disclosure, in certain embodiments, generally comprise a single amino acid chain that can, in some embodiments, comprise 4 “framework sequences” or FRs and 2 or 3 “complementary determining regions” or CDRs, preferably in a sequence FR1-CDR1-FR2- CDR2-FR3 -(optionally CDR3)-FR4. It should be clear that framework regions of nanobodies may also contribute to the binding of their antigens.

[0050] The terms “nanobody” and “SMVAD,” in their broadest sense, are not limited to a specific biological source or to a specific method of preparation. For example, the nanobodies of the disclosure can generally be obtained: (1) by isolating the VIIII domain of a naturally occurring heavy chain antibody; (2) by expression of a nucleotide sequence encoding a naturally occurring VHH domain; (3) by “humanization” of a naturally occurring VHH domain or by expression of a nucleic acid encoding a such humanized VHH domain (see, e.g., Sulea, Humanization of Camelid Single Domain Antibodies, Methods Mol Biol. 2022; 2446:299-312 and Vincke et al., General Strategy to Humanize a Camelid Single-domain Antibody and Identification of a Universal Humanized Nanobody Scaffold, The J. of Bio. Chem. Vol. 284, No. 5, pp. 3273-3284, January 30, 2009; both of which are herein incorporated in their entirities and particularly for methods of humanizing nanobodies); (4) by “camelization” of a naturally occurring VII domain from any animal species, and in particular from a mammalian species, such as from a human being, or by expression of a nucleic acid encoding such a camelized VH domain; (5) by “camelization” of a “domain Attorney Docket Number: CCF-43777.601 antibody” or “Dab,” as described in the art, or by expression of a nucleic acid encoding such a camelized VH domain; (6) by using synthetic or semi-synthetic techniques for preparing proteins, polypeptides or other amino acid sequences known per se; (7) by preparing a nucleic acid encoding a nanobody using techniques for nucleic acid synthesis known per se, followed by expression of the nucleic acid thus obtained; and / or (8) by any combination of one or more of the foregoing.

[0051] The amino acid residues of a nanobody are generally numbered according to the general numbering for VH domains given by Kabat et al., as applied to VHH domains from Camelids in the article of Riechmann and Muyldermans, J Immunol Methods 1999 Dec 10;23 l(l-2):25-38, herein incorporated by reference. According to this numbering, TRI of a Nanobody generally comprises the amino acid residues at positions 1-30, CDR1 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. It should be noted that it is well known in the art for VH domains and for VHH domains that 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 FR1 and vice versa, position 36 according to the

[0052] Kabat numbering corresponds to the start of FR2 and vice versa, position 66 according to the

[0053] Kabat numbering corresponds to the start of FR3 and vice versa, and position 103 according to the Kabat numbering corresponds to the start of FR4 and vice versa.

[0054] Nanobodies have a number of unique structural characteristics and functional properties which make isolated SMVADs, and proteins containing the same, highly advantageous for use as functional antigen-binding domains or proteins. In particular, and without being limited thereto, SMVADs, which have been “designed” by nature to functionally bind to an antigen without the presence of, and without any interaction with, a Attorney Docket Number: CCF-43777.601 light chain variable domain, can function as a single, relatively small, functional antigenbinding structural unit, domain or protein. This distinguishes the nanobodies 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 are composed of a VH domain covalently linked to a VL, domain).

[0055] In certain embodiments, the dual-antigen binding molecules comprise one or more of the nanobodies, SMVADs, or CDRs shown in SEQ ID NOS: 1-12 and / or variable regions or CDRs with one or more conservative or non-conservative amino acid changes in these SEQ ID NOS: 1-12, and nucleic acid sequences encoding SEQ ID NOs: l-12.

[0056] In carrying out site-directed mutagenesis of DNA encoding variable regions, the starting DNA is altered by first hybridizing an oligonucleotide encoding the desired mutation to a single strand of such starting DNA. After hybridization, a DNA polymerase is used to synthesize an entire second strand, using the hybridized oligonucleotide as a primer, and using the single strand of the starting DNA as a template. Thus, the oligonucleotide encoding the desired mutation is incorporated in the resulting double-stranded DNA.

[0057] PCR mutagenesis is also suitable for making amino acid sequence variants of the starting CDRs (see, e.g., Vallette et. al., (1989) Nucleic Acids Res. 17: 723-733, hereby incorporated by reference). Briefly, when small amounts of template DNA are used as starting material in a PCR, primers that differ slightly in sequence from the corresponding region in a template DNA can be used to generate relatively large quantities of a specific DNA fragment that differs from the template sequence only at the positions where the primers differ from the template.

[0058] Another method for preparing variants, cassette mutagenesis, is based on the technique described by Wells et al., (1985) Gene 34: 315-323, hereby incorporated by reference. The starting material is the plasmid (or other vector) comprising the starting CDR or variant region DNA to be mutated. The codon(s) in the starting DNA to be mutated are identified. There should be a unique restriction endonuclease site on each side of the identified mutation site(s). If no such restriction sites exist, they may be generated using the above-described oligonucleotide-mediated mutagenesis method to introduce them at appropriate locations in the starting polypeptide DNA. The plasmid DNA is cut at these sites to linearize it. A double- stranded oligonucleotide encoding the sequence of the DNA Attorney Docket Number: CCF-43777.601 between the restriction sites but containing the desired mutation(s) is synthesized using standard procedures, wherein the two strands of the oligonucleotide are synthesized separately and then hybridized together using standard techniques. This double-stranded oligonucleotide is referred to as the cassette. This cassette is designed to have 5' and 3’ ends that are compatible with the ends of the linearized plasmid, such that it can be directly ligated to the plasmid. This plasmid now contains the mutated DNA sequence.

[0059] Alternatively, or additionally, the desired amino acid sequence encoding a CDR variant, or variable region variant, can be determined, and a nucleic acid sequence encoding such amino acid sequence variant can be generated synthetically. Conservative modifications in the amino acid sequences of the CDRs or variable region may also be made. Naturally occurring residues are divided into classes based on common side-chain properties:

[0060] (1) hydrophobic: norleucine, met, ala, val, leu, ile;

[0061] (2) neutral hydrophilic: cys, ser, thr;

[0062] (3) acidic: asp, glu;

[0063] (4) basic: asn, gin, his, lys, arg;

[0064] (5) residues that influence chain orientation: gly, pro; and

[0065] (6) aromatic: trp, tyr, phe.

[0066] Conservative substitutions will entail exchanging a member of one of these classes for another member of the same class in a particular antibody, variable region, or CDR, such as in SEQ ID NOS: 1-12.

[0067] The CDRs of the present invention may be employed with any type of suitable framework. In some embodiments, the CDRs are used with fully human frameworks, or framework sub-regions. For example, the NCBI web site contains the sequences for known human framework regions. Examples of human VH sequences include, but are not limited to, VH1-18, VH1-2, VH1-24, VH1-3, VH1-45, VH1-46, VH1-58, VH1-69, VH1-8, VH2-26, VH2-5, VH2-70, VH3-1 1, VH3-13, VH3-15, VH3-16, VH3-20, VH3-21, VH3-23, VH3-30, VH3-33, VH3-35, VH3-38, VH3-43, VH3-48, VH3-49, VH3-53, VH3-64, VH3-66, VH3-7, VH3-72, VH3-73, VH3-74, VH3-9, VH4-28, VH4-31, VH4-34, VH4-39, VH4-4, VH4-59, VH4-61, VH5-51, VH6-1, and VH7-81. which are provided in Matsuda et al., (1998) J. Exp. Med. 188:1973-1975, that includes the complete nucleotide sequence of the human immunoglobulin chain variable region locus, herein incorporated by reference. Examples of human VK sequences include, but are not limited to, Al, A10, Al l, A14, A17, A18, A19, A2, A20, A23, A26, A27, A3, A30, A5, A7, B2, B3, LI, LIO, Li l, L12, L14, L15, L16, L18, L19, L2, L20, L22, L23, L24, L25, L4 / 18a, L5, L6, L8, L9, 01, Oi l, 012, 014, 018, 02, Attorney Docket Number: CCF-43777.601

[0068] 04, and 08, which are provided in Kawasaki et al., (2001) Eur. J. Immunol. 31:1017-1028; Schable and Zachau, (1993) Biol. Chem. Hoppe Seyler 374: 1001-1022; and Brensing- Kuppers et al., (1997) Gene 191 : 173-181, all of which are herein incorporated by reference. Examples of human VL sequences include, but are not limited to, V1-11, V1-13, V1-16, VI- 17, Vl-18, Vl-19, Vl-2, Vl-20, Vl-22, Vl-3, Vl-4, Vl-5, Vl-7, Vl-9, V2-1, V2-11, V2-13, V2-14, V2-15, V2-17, V2-19, V2-6, V2-7, V2-8, V3-2, V3-3, V3-4, V4-1, V4-2, V4-3, V4-4, V4-6, V5-1, V5-2, V5-4, and V5-6, which are provided in Kawasaki et al., (1997) Genome Res. 7:250-261 , herein incorporated by reference. Fully human frameworks can be selected from any of these functional germline genes. Generally, these frameworks differ from each other by a limited number of amino acid changes. These frameworks may be used with the CDRs described herein. Additional examples of human frameworks which may be used with the CDRs of the present invention include, but are not limited to, KOL, NEWM, REI, EU, TUR, TEI, LAY and POM (See, e.g., Kabat et al., (1991) Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA; and Wu et al., (1970), J. Exp. Med. 132:211-250, both of which are herein incorporated by reference).

[0069] In certain embodiments, the dual-antigen binding molecules of the present invention comprise antibodies or antibody fragments (e.g., comprising one or more of the CDRs described herein, such as in Figures IB and 1C), and a nanobody or antigen binding nanobody fragment. An antibody, or antibody fragment, of the present invention can be prepared, for example, by recombinant expression of immunoglobulin light and heavy chain genes in a host cell. For example, to express an antibody recombinantly, a host cell may be transfected with one or more recombinant expression vectors carrying DNA fragments encoding the immunoglobulin light and heavy chains of the antibody such that the light and heavy chains are expressed in the host cell and, preferably, secreted into the medium in which the host cell is cultured, from which medium the antibody can be recovered. Standard recombinant DNA methodologies may be used to obtain antibody heavy and light chain genes, incorporate these genes into recombinant expression vectors and introduce the vectors into host cells, such as those described in Sambrook, Fritsch and Maniatis (eds), Molecular Cloning; A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), Ausubel, F. M. et al. (eds.) Current Protocols in Molecular Biology, Greene Publishing Associates, (1989) and in U.S. Pat. No. 4,816,397 by Boss et al., all of which are herein incorporated by reference. Attorney Docket Number: CCF-43777.601

[0070] In certain antibodies, the antibody portion of the dual-antigen binding molecules herein, or fragments, thereof prepared herein have an IgG isotype constant regions as shown in Table 1 below.

[0071] TABLE 1

[0072] To express a dual-antigen binding molecule herein with one or more of the CDRs herein, DNA fragments encoding the light and / or heavy chain variable regions, SMVAD, are first obtained. These DNAs can be obtained by amplification and modification of germline light and heavy chain variable sequences (or SMVAD sequences) using the polymerase chain reaction (PCR).

[0073] Once the germline VH and VL fragments, and SMVAD sequences, are obtained, these sequences can be mutated to encode one or more of the CDR amino acid sequences disclosed herein (see, Figures IB and IB). The amino acid sequences encoded by the germline VH and VL, and SMVAD, DNA sequences may be compared to the CDRs sequence(s) desired to identify amino acid residues that differ from the germline sequences. Then the appropriate nucleotides of the germline DNA sequences are mutated such that the mutated germline sequence encodes the selected CDRs, using the genetic code to determine which nucleotide changes should be made. Mutagenesis of the germline sequences may be carried out by standard methods, such as PCR-mediated mutagenesis (in which the mutated nucleotides are incorporated into the PCR primers such that the PCR product contains the mutations) or site-directed mutagenesis. In other embodiments, the variable region is synthesized de novo (e.g., using a nucleic acid synthesizer).

[0074] Once DNA fragments encoding the desired VH and VL, and SMVAD, segments are obtained (e.g., by amplification and mutagenesis of germline VH and VL genes, or synthetic Attorney Docket Number: CCF-43777.601 synthesis, as described above), these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example to convert the variable region genes to full-length antibody chain genes, to Fab fragment genes or to a scFv gene. In these manipulations, a VL- or VH-encoding DNA fragment is operably linked to another DNA fragment encoding another polypeptide, such as an antibody constant region or a flexible linker. The isolated DNA encoding the VH region can be converted to a full-length heavy chain gene by operably linking the VH-encoding DNA to another DNA molecule encoding heavy chain constant regions (CHI , CH2 and CH3). The sequences of mouse and human heavy chain constant region genes are known in the art and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be, for example, an IgGl, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region. For a Fab fragment heavy chain gene, the VH-encoding DNA can be operably linked to another DNA molecule encoding only the heavy chain CHI constant region.

[0075] The isolated DNA encoding the VL region can be converted to a full-length light chain gene (as well as a Fab light chain gene) by operably linking the VL-encoding DNA to another DNA molecule encoding the light chain constant region, CL. The sequences of mouse and human light chain constant region genes are known in the art (see e.g., Kabat, E. A., et al., (1991) Sequences of Proteins of immunological Interest, Fifth Edition, U.S. Department of Health and Human Services. NIH Publication No. 91-3242) and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region.

[0076] To create a scFv gene, the VH- and VL-encoding DNA fragments may be operably linked to another fragment encoding a flexible linker, e.g., encoding the amino acid sequence (Gly4-Ser)3 (SEQ ID NO: 13), such that the VH and VL sequences can be expressed as a contiguous single-chain protein, with the VL and VH regions joined by the flexible linker (see e.g., Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; and McCafferty et al., (1990) Nature 348:552-554), all of which are herein incorporated by reference).

[0077] To express the dual-antigen binding molecules herein, DNAs encoding SMVADs, partial or full-length light and heavy chains, (e.g. obtained as described above), may be inserted into expression vectors such that the genes are operably linked to transcriptional and translational control sequences. In this context, the term "operably linked" is intended to mean that an antibody or nanobody gene is ligated into a vector such that transcriptional and translational control sequences within the vector serve their intended function of regulating the transcription and translation of the antibody gene. The expression vector and expression Attorney Docket Number: CCF-43777.601 control sequences are generally chosen to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene can be inserted into separate vectors or, more typically, both genes are inserted into the same expression vector.

[0078] The antibody and nanobody genes may be inserted into the expression vector by standard methods (e.g., ligation of complementary restriction sites on the antibody gene fragment and vector, or blunt end ligation if no restriction sites are present). Prior to insertion of the light or heavy chain sequences, the expression vector may already carry antibody constant region sequences. For example, one approach to converting the VH and VL sequences to full-length antibody genes is to insert them into expression vectors already encoding heavy chain constant and light chain constant regions, respectively, such that the VH segment is operably linked to the CH segment(s) within the vector and the VL segment is operably linked to the CL segment within the vector. Additionally, or alternatively, the recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody chain from a host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).

[0079] In addition to the antibody chain genes and nanobody genes, the recombinant expression vectors of the disclosure may carry regulatory sequences that control the expression of the antibody chain genes and nanobody genes in a host cell. The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody chain genes. Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990), herein incorporated by reference. It will be appreciated by those skilled in the art that the design of the expression vector, including the selection of regulatory sequences may depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. In certain embodiments, regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and / or enhancers derived from cytomegalovirus (CMV) (such as the CMV promoter / enhancer), Simian Virus 40 (SV40) (such as the SV40 promoter / enhancer), adenovirus, (e.g., the adenovirus major late promoter (AdMLP)) and polyoma virus. For further description of viral regulatory elements, and sequences thereof, see e.g., U.S. Pat. No. 5,168,062 by Stinski, U.S. Pat. No. 4,510,245 by Attorney Docket Number: CCF-43777.601

[0080] Bell et al. and U.S. Pat. No. 4,968,615 by Schaffner et al., all of which are herein incorporated by reference.

[0081] In addition to the antibody chain genes, and nanobody genes, and regulatory sequences, the recombinant expression vectors of the invention may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see e.g., U.S. Pat. Nos. 4,399,216, 4,634.665 and 5,179,017, all by Axel et al.). For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, on a host cell into which the vector has been introduced. Selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr- host cells with methotrexate selection / amplification) and the neomycin gene (for G418 selection).

[0082] For expression of the light and heavy chains, the expression vector(s) encoding the heavy and light chains may be transfected into a host cell by standard techniques. The various forms of the term "transfection" are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, e.g., electroporation, calcium-phosphate precipitation, DEAE-dextran transfection and the like.

[0083] In certain embodiments, the expression vector used to express the dual-antigen binding molecules of the present invention are viral vectors, such as retro-viral vectors. Such viral vectors may be employed to generate stably transduced cell lines (e.g. for a continues source of the molecules). In some embodiments, the GPEX gene product expression technology (from Catalent, Somerset, NJ) is employed to generate the dual-antigen binding molecules (and stable cell lines expressing the dual-antigen binding molecules). In particular embodiments, the expression technology described in W00202783 and W00202738 to Bieck et al. (both of which are herein incorporated by reference in their entireties) is employed.

[0084] Mammalian host cells for expressing the dual-antigen binding molecules of the invention include, for example, cells Chinese Hamster Ovary (CHO cells) (including dhfr- CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216- 4220, used with a DHFR selectable marker, e.g., as described in R. J. Kaufman and P. A. Sharp (1982) Mol. Biol. 159:601-621), NSO myeloma cells, COS cells and SP2 cells. In other embodiments, the host cells express GnT III as described in WO9954342 and U.S. Pat. Pub. 20030003097, both herein incorporated by reference, such that expressed dual-antigen binding molecules have increased ADCC activity. Attorney Docket Number: CCF-43777.601

[0085] In certain embodiments, the dual-antigen binding molecules of the present invention are useful for immunoassays which detect or quantify human CFD and C2 in a sample (e.g., a purified blood sample from a subject). In some embodiments, an immunoassay for both CFD and C2 typically comprises incubating a biological sample in the presence of a detectably labeled dual-antigen binding molecules capable of selectively binding to both CFD and C2, and detecting the labeled dual-antigen binding molecule which is bound in a sample. Various clinical assay procedures are well known in the art.

[0086] The present disclosure provides immunoassay methods for determining the presence, amount or concentration of human CFD and C2 in a test sample. Any suitable assay known in the art can be used in such a method. Examples of such assays include, but are not limited to, immunoassay, such as sandwich immunoassay (e.g., monoclonal-polyclonal sandwich immunoassays, including radioisotope detection (radioimmunoassay (RIA)) and enzyme detection (enzyme immunoassay (EIA) or enzyme-linked immunosorbent assay (ELISA) (e.g., Quantikinc ELISA assays, R&D Systems, Minneapolis, Minn.)), competitive inhibition immunoassay (e.g., forward and reverse), fluorescence polarization immunoassay (FPIA), enzyme multiplied immunoassay technique (EMU ), an ARCH1TEC T assay (ABBOTT), a bioluminescence resonance energy transfer (BRET), and homogeneous chemiluminescent assay, etc.

[0087] In certain embodiments, provided here are kits for the detection of CFD and C2 that include a human CFD and human C2 and the dual- antigen detection molecules herein. Such kits may include any of the immunodiagnostic reagents described herein and may further include instructions for the use of the immunodiagnostic reagents in immunoassays for determining the presence of human CFD and C2 in a test sample. The kits may also include other reagents required to conduct a diagnostic assay or facilitate quality control evaluations, such as buffers, salts, enzymes, enzyme co-factors, substrates, detection reagents, and the like. Other components, such as buffers and solutions for the isolation and / or treatment of a test sample (e.g., pretreatment reagents), also can be included in the kit. The kit can additionally include one or more other controls. One or more of the components of the kit can be lyophilized, in which case the kit can further comprise reagents suitable for the reconstitution of the lyophilized components. The various components of the kit may be provided in suitable containers as necessary, e.g., a microtiter plate. The kit can further include containers for holding or storing a sample (e.g., a container or cartridge for a sample). Where appropriate, the kit optionally also can contain reaction vessels, mixing vessels, and other components that facilitate the preparation of reagents or the test sample. The kit can Attorney Docket Number: CCF-43777.601 also include one or more instrument for assisting with obtaining a test sample, such as a syringe, pipette, forceps, measured spoon, or the like.

[0088] All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in chemistry, medicine, and molecular biology or related fields are intended to be within the scope of the following claims.

Claims

Attorney Docket Number: CCF-43777.601CLAIMS:We claim:

1. A composition comprising: a dual-antigen binding molecule, or one or more nucleic acid molecules encoding said dual-antigen binding molecule, wherein said dual-antigen binding molecule comprises: i) a human complement factor D (FD) binding region comprising:A) an antibody light chain comprising a light chain variable region,B) an antibody heavy chain comprising a heavy chain variable region and at least one heavy chain constant region: ii) a human complement component 2 (C2) binding region comprising:A) a first single monomeric variable antibody domain (SMVAD1), andB) optionally a second single monomeric variable antibody domain (SMVAD2) which, if present, is joined to said SMVAD1 by a second linker; and iii) a first linker attached to said at least one heavy constant region and to said SMVAD1.

2. The composition of claim 1 , wherein said light chain variable region comprises at least one of the following: i) a CDR1 amino acid sequence comprising SEQ ID NO:9, or SEQ ID NO:9 with one conservative amino acid change; ii) a CDR2 amino acid sequence comprising SEQ ID NOTO, or SEQ ID NO: 10 with one conservative amino acid change; iii) a CDR3 amino acid sequence comprising SEQ ID NO: 11, or SEQ ID NO: 11 with one conservative amino acid change.

3. The composition of claim 2, wherein said light chain variable region comprises four Framework regions, wherein said four Framework regions are camelid, humanized, or human Framework regions.

4. The composition of claim 2, wherein said antibody light chain comprises a light chain constant region.Attorney Docket Number: CCF-43777.6015. The composition of claim 1, wherein said heavy chain variable regions comprises at least one of the following: i) a CDR1 amino acid sequence comprising SEQ ID NO:2, or SEQ ID NO:2 with one conservative amino acid change; ii) a CDR2 amino acid sequence comprising SEQ ID NO:3, or SEQ ID NO:3 with one conservative amino acid change; iii) a CDR3 amino acid sequence comprising SEQ ID NO:4, or SEQ ID NO:4 with one conservative amino acid change.

6. The composition of claim 5, wherein said heavy chain variable region comprises four Framework regions, wherein said four Framework regions are camelid, humanized, or human Framework regions.

7. The composition of claim 1, wherein said SMVAD1 and / or SMVAD 2 comprises at least one of the following: i) a CDR1 amino acid sequence comprising SEQ ID NO:5, or SEQ ID NO:5 with one conservative amino acid change; ii) a CDR2 amino acid sequence comprising SEQ ID NO:6, or SEQ ID NO:6 with one conservative amino acid change; iii) a CDR3 amino acid sequence comprising SEQ ID NO:7, or SEQ ID NO:7 with one conservative amino acid change.

8. The composition of claim 7, wherein said SMVAD1 and SMVAD2 further comprise four Framework regions, wherein said four Framework regions are camelid, humanized, or human Framework regions.

9. The composition of claim 1, wherein said SMVAD1 and / or SMVAD 2 comprises SEQ ID NO:12, or SEQ ID NO:12 with one conservative amino acid change.

10. The composition of claim 1, wherein said at least one heavy chain constant region comprises three heavy chain constant regions.Attorney Docket Number: CCF-43777.60111. The composition of claim 1, wherein said composition comprises two of said dualantigen binding molecules such that the at least one heavy chain constant region on each are aligned with each other.

12. The composition of claim 1, further comprising a physiologically tolerable buffer.

13. The composition of claim 1, wherein said composition comprises said one or more nucleic acid molecules, and optionally the composition further comprises an expression vector, and wherein said first and / or second nucleic acid sequences are present in said expression vector.

14. The composition of claim 1, wherein said composition comprises said dual-antigen binding molecule.

15. A method of treating or preventing a complement- related disease or condition comprising: treating a subject with a composition comprising said dual-antigen binding molecule, or an expression vector comprising said one or more nucleic acid molecules encoding said dual-antigen binding molecule of any of Claims 1-14, and wherein said subject has, or is suspected to develop, a complement-related disease or condition.

16. The method of claim 15, wherein said complement-related disease comprises a dysregulated complement activation disease.

17. The method of claim 15, wherein said complement-related disease comprises myasthenia gravis or atypical hemolytic uremic syndrome.

18. The method of claim 15, wherein said complement- related disease comprises paroxysmal nocturnal hemoglobinuria (PNH) or and autoimmune hemolytic anemia (AIHA).

19. The method of claim 15, wherein said composition comprises said expression vector, and wherein said first and / or second nucleic acid sequences are present in said expression vector.Attorney Docket Number: CCF-43777.60120. The method of claim 15, wherein said composition comprises said dual-antigen binding molecule.

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