MODIFIED IgG1 Fc DOMAINS AND ANTI-CD40 DOMAIN ANTIBODY FUSIONS THEREWITH

Abstract

To provide an antibody polypeptide that specifically binds a human CD40.SOLUTION: An antibody polypeptide comprises a human IgG1 Fc domain polypeptide, wherein the human IgG1 Fc domain polypeptide comprises a mutation at Kabat position 238 that reduces binding to FC-gamma-receptors, proline 238 (P238) is mutated to lysine, the antibody polypeptide binds CD40, and the antibody polypeptide comprises reduced binding to Fcg receptor hCD16b-NA2 (FcgRIIIb-NA2).SELECTED DRAWING: None

Description

[Technical Field]

[0001] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format, which is incorporated herein by reference in its entirety. The ASCII copy, created on May 23, 2018, is named 200896_0014_00_WO_ST25.txt and is 245,881 bytes in size.

[0002] Technical Field The present invention provides a modified IgG1 Fc domain with reduced binding to Fc gamma receptors. It also provides an antibody polypeptide comprising an anti-CD40 single variable domain and a modified Fc domain. The antibody polypeptide binds to CD40, does not exhibit CD40 agonist activity, does not activate immature dendritic cells, and has improved biophysical properties suitable for development as a therapeutic agent. It also provides compositions comprising the antibody polypeptide, methods of use for treating diseases involving CD40 activity, and uses in preparing medicaments for treating diseases involving CD40 activity. [Background technology]

[0003] CD40 is a costimulatory molecule belonging to the tumor necrosis factor (TNF) receptor superfamily that is present on antigen-presenting cells (APCs), including dendritic cells, B cells, and macrophages. H Upon binding to its ligand CD154 (CD40L) on cells, APCs are activated. CD40-mediated APC activation is involved in various immune responses, including cytokine production, upregulation of costimulatory molecules (such as CD86), and enhancement of antigen presentation and B cell proliferation. CD40 can also be expressed by endothelial cells, smooth muscle cells, fibroblasts, and epithelial cells.

[0004] CD40 activation has also been implicated in various undesired T cell responses, such as those associated with autoimmunity, transplant rejection, or allergic reactions. One strategy for controlling undesired T cell responses is to target CD40 with antagonistic antibodies. For example, the monoclonal antibody HCD122 (lucatumumab, formerly known as Chiron1212) is currently in clinical trials for the treatment of certain CD40-mediated inflammatory diseases. See "Study of HCD122 (Lucatumumab) and Bendamustine Combination Therapy in CD40" at: clinicaltrialsfeeds.org / clinical-trials / show / NCT01275209 (last updated January 11, 2011). + See "Rituximab - Refractory Follicular Lymphoma", Clinical Trials Feeds. However, monoclonal antibodies can exhibit agonist activity. For example, the utility of the anti-CD40 antibody Chi220 is limited by its weak stimulatory capacity. See Adams, et al., 2005, "Development of a chimeric anti-CD40 monoclonal antibody that synergizes with LEA29Y to prolong islet allograft survival", J. Immunol. 174: 542-50.

[0005] There remains a need for therapies that modulate CD40 activation in the treatment and / or prevention of immune diseases. Summary of the Invention

[0006] A human IgG1 Fc domain polypeptide is provided comprising a Kabat mutation at position 238 that reduces binding to Fc gamma receptors, wherein the proline at position 238 (P238) is mutated to one of the residues selected from lysine, serine, alanine, arginine, and tryptophan. The IgG1 Fc may comprise the amino acid sequence of SEQ ID NO:65.

[0007] Provided is a human IgG1 Fc domain polypeptide comprising a lysine substituted at Kabat position 238. An exemplary amino acid sequence of a human IgG1 Fc domain polypeptide is as follows: EPKSCDKTHTCPPCPAPELLGG K SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (IgG1a-P238K (-C-terminal lysine); SEQ ID NO: 134), EPKSCDKTHTCPPCPAPELLGG K SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (IgG1a-P238K; SEQ ID NO: 66), EPKSCDKTHTCPPCPAPELLGG KSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (IgG1f-P238K(-C-terminal lysine); SEQ ID NO: 135), and EPKSCDKTHTCPPCPAPELLGG K SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (IgG1f-P238K; SEQ ID NO: 67).

[0008] Provided is (A) a heterologous polypeptide, and (B) a fusion polypeptide comprising the Fc domain described above.

[0009] Further provided are antibody polypeptides comprising: (1) a single variable domain comprising: (a) a CDR1 region comprising the amino acid sequence of SEQ ID NO: 1, or a CDR1 region that differs from the CDR1 region of SEQ ID NO: 1 by at most two amino acids, (b) a CDR2 region comprising the amino acid sequence of SEQ ID NO: 2, or a CDR2 region that differs from the CDR2 region of SEQ ID NO: 2 by at most three amino acids, and (c) a CDR3 region comprising the amino acid sequence of SEQ ID NO: 3, or a CDR3 region that differs from the CDR3 region of SEQ ID NO: 3 by at most six amino acids, wherein the single variable domain binds to CD40; and (2) an Fc domain that is a human IgG1 Fc domain polypeptide comprising a Kabat mutation at position 238 that reduces binding to Fc gamma receptors, wherein the proline at position 238 (P238) is mutated to one of a residue selected from lysine, serine, alanine, arginine, and tryptophan. The single variable domains of the antibody polypeptides described herein antagonize at least one activity of CD40. The antibody polypeptides described herein have improved stability compared to a reference polypeptide having the same single variable domain sequence and fused to a wild-type IgG1 Fc domain. Provided are antibody polypeptides comprising: (1) the single variable domain described above, wherein the human IgG1 Fc domain has a lysine substitution at Kabat position 238. An exemplary amino acid sequence of a human IgG1 Fc domain polypeptide is as follows: EPKSCDKTHTCPPCPAPELLGG K SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (IgG1a-P238K (-C-terminal lysine); SEQ ID NO: 134), EPKSCDKTHTCPPCPAPELLGG KSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (IgG1a-P238K; SEQ ID NO: 66), EPKSCDKTHTCPPCPAPELLGG K SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (IgG1f-P238K(-C-terminal lysine); SEQ ID NO: 135), and EPKSCDKTHTCPPCPAPELLGG K SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (IgG1f-P238K; SEQ ID NO: 67).

[0010] Further provided is the above-described antibody polypeptide, wherein (a) the CDR1 region consists of the sequence X1-Tyr-Glu-Y1-Trp (SEQ ID NO: 4), where X1 is Asp or Gly and Y1 is Met or Leu; (b) the CDR2 region consists of the sequence Ala-Ile-Asn-Pro-X2-Gly-Y2-Z2-Thr-Tyr-Tyr-Ala-Asp-Ser-Val-A2-Gly (SEQ ID NO: 5), where X2 is Gln, Tyr, His, Trp, or Ala, Y2 is Thr, Asn, Gly, Ser, or Gln, and Z2 is Arg, L (c) the CDR3 region consists of the sequence X3-Pro-Y3-Z3-A3-B3-C3 (SEQ ID NO: 6), where X3 is Leu, Pro or Glu, Y3 is Phe, Gln, Thr, Met or Tyr, Z3 is Arg, Tyr, Pro, Leu, Thr, Ile, Phe, Met or Ser, A3 is Phe or Tyr, B3 is Ser, Gln, His, Asp, Lys, Glu or Gly, and C3 is Asp, Tyr, Glu or Ser.

[0011] Further provided is the above-mentioned antibody polypeptide, wherein (a) the CDR1 region consists of the amino acid sequence of SEQ ID NO: 1 (CDR1 of 3h-56-269), (b) the CDR2 region consists of the amino acid sequence of SEQ ID NO: 2 (CDR2 of 3h-56-269), and (c) the CDR3 region consists of the amino acid sequence of SEQ ID NO: 3 (CDR3 of 3h-56-269). Further provided is the above-mentioned antibody polypeptide, wherein the amino acid sequence of the single variable domain is set forth in SEQ ID NO: 41 (= the 3h-56-269 sequence).

[0012] An antibody polypeptide is provided that comprises or consists of the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGFTFRDYEMWWVRQAPGKGLERVSAINPQGTRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKLPFRFSDRGQGTLVTVSS ASTEPKSCDKTHTCPPCPAPELLGG K SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 136), or EVQLLESGGGLVQPGGSLRLSCAASGFTFRDYEMWWVRQAPGKGLERVSAINPQGTRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKLPFRFSDRGQGTLVTVSS AST EPKSCDKTHTCPPCPAPELLGG K SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 70).

[0013] An antibody polypeptide is provided that comprises or consists of the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGFTFRDYEMWWVRQAPGKGLERVSAINPQGTRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKLPFRFSDRGQGTLVTVSS AST EPKSCDKTHTCPPCPAPELLGG KSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 137), or EVQLLESGGGLVQPGGSLRLSCAASGFTFRDYEMWWVRQAPGKGLERVSAINPQGTRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKLPFRFSDRGQGTLVTVSS AST EPKSCDKTHTCPPCPAPELLGG K SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 71).

[0014] Nucleic acids encoding any of the human IgG1 Fc domain polypeptides, fusion polypeptides, or antibody polypeptides of the present disclosure are further provided. Also provided are expression vectors containing the nucleic acid molecules. Cells transformed with the expression vectors are provided.

[0015] A pharmaceutical composition is provided which comprises the above-described antibody polypeptide and a pharmaceutically acceptable carrier.

[0016] Also provided is a method for treating or preventing an immune disease in a subject, comprising administering to the subject the above-described antibody polypeptide. The immune disease may be selected from the group consisting of Addison's disease, allergy, anaphylaxis, ankylosing spondylitis, asthma, atherosclerosis, atopic allergy, autoimmune diseases of the ear, autoimmune diseases of the eye, autoimmune hepatitis, autoimmune parotitis, bronchial asthma, coronary heart disease, Crohn's disease, diabetes, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hemolytic anemia, idiopathic thrombocytopenic purpura, inflammatory bowel disease, immune response to recombinant products (e.g., factor VII in hemophilia), systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjögren's syndrome, spondyloarthropathy, thyroiditis, transplant rejection, vasculitis, and ulcerative colitis. [Brief explanation of the drawings]

[0017] [Figure 1]Figure 1 (including Figures 1A and 1B) shows the amino acid sequences of representative antibody polypeptides useful in the present disclosure. Figure 1A shows the amino acid sequence (SEQ ID NO:70) of an antibody polypeptide fusion of the single variable domain antibody BMS3h-56-269 (SEQ ID NO:41) with an Fc domain (IgG1a-P238K; SEQ ID NO:66). The amino acid sequence of the Fc domain (SEQ ID NO:66) is shown in italics; the underlined and italicized residue at position 23 (corresponding to Kabat position 238) is a proline to lysine mutation. Figure 1B shows the amino acid sequence (SEQ ID NO:71) of an antibody polypeptide fusion of the single variable domain antibody BMS3h-56-269 (SEQ ID NO:41) with another Fc domain (IgG1f-P238K; SEQ ID NO:67). The amino acid sequence of the Fc domain (SEQ ID NO:67) is shown in italics. In both Figures 1A and 1B, the three complementarity determining regions of the single variable domain, CDR1 (SEQ ID NO: 1), CDR2 (SEQ ID NO: 2), and CDR3 (SEQ ID NO: 3), are underlined. The amino acids of the four framework regions, FR1 (SEQ ID NO: 42), FR2 (SEQ ID NO: 44), FR3 (SEQ ID NO: 47), and FR4 (SEQ ID NO: 54), are not underlined. The amino acid sequence of the linker, AST (SEQ ID NO: 57), is double underlined.

[0018] [Figure 2A] Figure 2A shows iDC activation data for up to nine iDC donors with various concentrations of dAb-Fc fusions. Dose response of BMS-986090 (anti-CD40 dAb fused to IgG4 Fc), CD40L (soluble CD40L trimer (via an isoleucine zipper trimerization motif)), and mAb 134-2141 (agonistic anti-CD40 antibody) on CD86 expression. ChiL6-IgG4 (control-L6): negative control. Unstim: iDCs only (unstimulated). Concentrations are shown in μg / ml. [Figure 2B]Figure 2B shows iDC activation data for up to nine iDC donors at various concentrations of dAb-Fc fusions. Dose responses of BMS-986090 (anti-CD40 dAb fused to IgG4 Fc), CD40L (soluble CD40L trimer (via an isoleucine zipper trimerization motif)), and mAb 134-2141 (agonistic anti-CD40 antibody) on ICAM-1 expression. ChiL6-IgG4 (control-L6): negative control. Unstim: iDCs only (unstimulated). Concentrations are shown in μg / ml. [Figure 2C] Figure 2C shows iDC activation data for up to nine iDC donors at various concentrations of dAb-Fc fusions. Dose responses of BMS-986090 (anti-CD40 dAb fused to IgG4 Fc), CD40L (soluble CD40L trimer (via an isoleucine zipper trimerization motif)), and mAb 134-2141 (agonistic anti-CD40 antibody) on IL-6 release. ChiL6-IgG4 (control-L6): negative control. Unstim: iDCs only (unstimulated). Concentrations are shown in μg / ml. [Figure 2D] Figure 2D shows iDC activation data for up to nine iDC donors at various concentrations of dAb-Fc fusions. Dose responses of BMS-986090 (anti-CD40 dAb fused to IgG4 Fc), CD40L (soluble CD40L trimer (via an isoleucine zipper trimerization motif)), and mAb 134-2141 (agonistic anti-CD40 antibody) on TNF-alpha release. ChiL6-IgG4 (control-L6): negative control. Unstim: iDCs only (unstimulated). Concentrations are shown in μg / ml. [Figure 2E] Figure 2E shows iDC activation data for up to nine iDC donors at various concentrations of dAb-Fc fusions. Figure 2E shows a comparison of iDCs from up to nine donors treated with 100 μg / ml BMS-986090 or 3h-59-269-aba (dAb-IgG1 fusion).

[0019] [Figure 3]Figure 3 (including Figures 3A-3D) shows that CD32-mediated crosslinking / clustering of 3h-59-269-IgG4 increases iDC activation, as measured by CD86 expression (Figure 3A), ICAM expression (Figure 3B), and cytokine release (IL-6 in Figure 3C and TNF-alpha in Figure 3D). iDCs treated at the indicated concentrations (µg / ml) in solution or with crosslinking ("x-link") are shown; crosslinking refers to the addition of CD32-expressing CHO cells. ChiL6-IgG4 serves as a negative control.

[0020] [Figure 4] Figure 4 shows data from an iDC activation assay using anti-CD40 dAbs containing IgG4, IgG1.1f, IgG1.3f, and CT Fc tails. L6-IgG4 (ChiL6-IgG4) serves as a negative control; the agonist anti-CD40 mAb 1234-2141 is a positive control. iDCs were treated with the indicated concentrations (µg / ml). Addition of CD32-expressing CHO cells to iDC cultures (right panel) results in a significant increase in cytokine release and upregulation of activation markers for all fusion proteins except 3h-59-269-CT.

[0021] [Figure 5] Figure 5 (including Figures 5A-5E) shows DSC thermogram data for dAb-Fc molecules. Figure 5A) 3h56-269-IgG4.1. Figure 5B) 3h56-269-CT. Figure 5C) 3h56-269-IgG1-D265A. Figure 5D) 3h56-269-IgG1.1f. Figure 5E) 3h56-269-IgG1.3f. In each panel, the thick line indicates the thermogram data, and the thin line indicates the simplest best fit.

[0022] [Figure 6]Figure 6 (including Figures 6A-6F) shows the icIEF data for the dAb-Fc molecules: Figure 6A) 3h56-269-IgG4.1; Figure 6B) 3h56-269-CT; Figure 6C) 3h56-269-CT (produced from UCOE-CHO cells); Figure 6D) 3h56-269-IgG1-D265A; Figure 6E) 3h56-269-IgG1.1f; Figure 6F) 3h56-269-IgG1.3f. pI markers are indicated in panel A at pI 5.85 and pI 10.10.

[0023] [Figure 7] FIG. 7 shows SPR sensorgram data for the capture of 7 μg / ml hCD64-His with binding of the four 1F4 antibodies at 1 μM.

[0024] [Figure 8] FIG. 8 shows the icIEF data of 13 1F4 monoclonal antibodies with different Fc domains, with pI values ​​indicated.

[0025] [Figure 9] Figure 9 shows iDC activation data for 3h-59-269-IgG1-P238K and 3h-59-269-IgG1-N297A. L6-IgG4 (ChiL6-IgG4) serves as a negative control; BMS-986090 (3h-59-269-IgG4) is a positive control. iDCs were treated with the antibodies at the concentrations (μg / ml) indicated in the figure. Addition of CD32-expressing CHO cells to iDC cultures (data on the left side of each graph, indicated as "+CD32 CHO") resulted in a significant increase in cytokine release and upregulation of activation markers for the positive control 3h-59-269-IgG4, but not for dAbs fused to IgG1 Fc tails with single mutations at positions P238 or N297.

[0026] [Figure 10]Figure 10 shows iDC activation by anti-CD40 domain antibody-Fc fusion proteins with various Fc tails. L6-IgG4 (ChiL6-IgG4) serves as a negative control; agonist mAb 1234-2141 and BMS-986090 (3h-59-269-IgG4) are positive controls. iDCs were treated with the antibodies at the concentrations (μg / ml) indicated in the figure. iDC activation was observed with all fusion proteins except for fusions containing the P238K or N297A mutations, and was increased by the addition of CD32-expressing CHO cells (shown on the left side of each graph as "+CD32 CHO").

[0027] [Figure 11] Figure 11 (including Figures 11A-11D) shows DSC thermogram data for dAb-Fc molecules. Figure 11A) 3h56-269-IgG1a-C220S, C226A, C229A, P238S. Figure 11B) 3h56-269-IgG1a-C220S, C226A, C229A, P238K. Figure 11C) 3h56-269-IgG1a-C220S, P238K. Figure 11D) 3h56-269-IgG1f-C220S, N297A. DETAILED DESCRIPTION OF THE INVENTION

[0028] In accordance with this detailed description, the following abbreviations and definitions apply. It should be noted that, as used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to "an antibody" includes a plurality of such antibodies, a reference to "the dosage" includes a reference to one or more dosages and equivalents thereof known to those skilled in the art, and so forth.

[0029] As used herein, the term "about" is understood by those of ordinary skill in the art and will vary to some extent depending on the context in which it is used. Generally, about encompasses a range of values ​​of plus / minus 10% of the referenced value.

[0030] It is understood that every whole or partial integer between the ranges stated herein is included herein.

[0031] Abbreviations used herein: APC antigen presenting cells CD54 also known as ICAM-1 CDR Complementarity Determining Region C H constant heavy chain C L constant light chain CHO cells Chinese hamster ovary cells dAb domain antibodies DSC Differential Scanning Calorimetry FcgR Fc gamma receptor (interchangeable with FcγR) FR Framework Area FSB Fetal Bovine Serum GM-CSF Granulocyte-macrophage colony-stimulating factor iDC immature dendritic cells icIEF Imaged capillary isoelectric focusing IFN Interferon IgG immunoglobulin G IL-4 Interleukin 4 IL-6 Interleukin 6 mAb Monoclonal antibody mg milligram ml or mL milliliter ng nanogram pI isoelectric point SPR Surface Plasmon Resonance TNF tumor necrosis factor μg microgram V L Variable light chain V H variable heavy chain Additional abbreviations and definitions are provided herein.

[0032] 1. Fc domain The carboxy-terminal "half" of the heavy chain defines a constant region (Fc) primarily responsible for effector function. As used herein, the term "Fc domain" refers to the constant region of the heavy chain as defined by Kabat et al., Sequences of Immunological Interest, 5 th ed., US Dept. Health & Human Services, Washington, DC (1991). The Fc domain disclosed herein is derived from human IgG, and more specifically, from the human IgG1 Fc region. The human IgG1 Fc domain contains a mutation at Kabat position 238. The mutation replaces the proline at position 238 (P238) with an amino acid selected from lysine (K), serine (S), alanine (A), arginine (R), and tryptophan (W); or an amino acid selected from lysine and serine; or an amino acid selected from lysine.

[0033] An exemplary consensus sequence for an IgG1 Fc domain is: EPKSCDKTHTCPPCPAPELLGG X SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP QVYTLPPSR(D / E)E(L / M)TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Z (SEQ ID NO: 65), where X is K, S, A, R or W, Z is K or absent. In this sequence, position 23 (underlined X ) corresponds to Kabat's 238th position.

[0034] Exemplary IgG Fc domain sequences are in Table 1. Mutated residues are underlined. SEQ ID NOs: 134 and 135 are further exemplary IgG Fc domain sequences. [Table 1]

[0035] Although human IgG heavy chain genes encode a C-terminal lysine, the lysine is often absent from endogenous antibodies due to cleavage during circulation. When expressed in mammalian cell culture, antibodies having IgG heavy chains containing a C-terminal lysine may have varying levels of C-terminal lysine present (Cai et al., 2011, Biotechnol Bioeng. 108(2):404-12). Therefore, the C-terminal lysine of any IgG heavy chain Fc domain disclosed herein may be omitted. See, for example, SEQ ID NOs: 66 and 134, and SEQ ID NOs: 67 and 135. Similarly, the C-terminal lysine of SEQ ID NOs: 68 and 69 may optionally be absent.

[0036] The mutant IgG1 Fc domain exhibits reduced binding to Fc gamma receptors. Advantageously, reduced binding to Fc gamma receptors reduces or prevents iDC activation, as measured by at least one of the following: 1) release of cytokines IL-6 and / or TNF alpha; and 2) upregulation of cell surface expression of CD86 and / or CD54. Reduced binding to Fc gamma receptors is also believed to reduce or prevent clustering / crosslinking of FcgRs on immature dendritic cells. Furthermore, the mutant IgG1 Fc domain may contribute to the thermal stability and homogeneity of antibody polypeptides containing the mutant IgG1 Fc domain.

[0037] 2. Antibody Polypeptides Containing Mutant IgG1 Fc Domains The present disclosure includes fusion polypeptides comprising mutated IgG1 Fc domains.

[0038] 2.1. Heterologous Polypeptides The present disclosure includes fusion polypeptides of a heterologous polypeptide of the present disclosure and a mutant IgG1 Fc domain. The heterologous polypeptide may comprise or consist of a heavy chain variable domain. The carboxyl terminus of the heavy chain variable domain may be linked or fused to the amino terminus of the Fc domain. Alternatively, the carboxyl terminus of the heavy chain variable domain may be linked or fused to the amino terminus of a linker amino acid sequence, which is itself fused to the amino terminus of the Fc domain. Alternatively, the carboxyl terminus of the heavy chain variable domain may be linked or fused to the amino terminus of a CH1 domain, which is itself fused to the Fc domain. The fusion polypeptide may include a hinge region, in whole or in part, between the CH1 and CH2 domains. For example, an amino acid linker sequence is present between the heavy chain variable domain and the Fc domain.

[0039] 2.2. Domain Antibodies The present disclosure further includes single variable domains fused to an Fc domain (domain antibodies). A "domain antibody" (dAb) is a single variable domain (V) capable of specific and monovalent binding to an antigen (such as CD40). L or V H ). The carboxyl terminus of the single variable domain may be linked or fused to the amino terminus of an Fc CH2 domain. Alternatively, the carboxyl terminus of the single variable domain may be linked or fused to the amino terminus of a linker amino acid sequence, which is itself fused to the amino terminus of the Fc domain. Alternatively, the carboxyl terminus of the single variable domain may be linked or fused to the amino terminus of a CH1 domain, which is itself fused to the Fc CH2 domain. The protein may comprise a hinge region, in whole or in part, between the CH1 and CH2 domains. For example, an amino acid linker sequence is present between the single variable domain and the Fc domain. Also provided is an antibody polypeptide that is a fusion polypeptide comprising an anti-human CD40 domain antibody and a modified human Fc domain. Optionally, the antibody polypeptide further comprises an amino acid linker interposed between the domain antibody and the Fc domain. An exemplary antibody polypeptide is shown in Figure 1.

[0040] 2.2.1. Anti-CD40 Domain Antibodies Antibody polypeptides of the present disclosure include domain antibodies that specifically bind to human CD40 and do not exhibit CD40 agonist activity. A "domain antibody" (dAb) is an antibody that contains a single variable domain (V) that is capable of specifically and monovalently binding to an antigen (such as CD40). L or V H ) Domain antibodies are "V H They contain a "domain" and are human. Bivalent anti-CD40 antibodies are believed to exhibit agonist activity due to their ability to crosslink CD40 molecules bound on cell surfaces. Without being limited to a particular theory, it is believed that monovalent dAbs do not crosslink CD40 and therefore do not activate CD40.

[0041] CD40 is also known as B-cell surface antigen CD40, Bp50, CD40L receptor, CDw40, CDW40, MGC9013, p50, TNFRSF5, and tumor necrosis factor receptor superfamily member 5. "Human CD40" refers to a CD40 comprising the following amino acid sequence: MVRLPLQCVL WGCLLTAVHP EPPTACREKQ YLINSQCCSL CQPGQKLVSD CTEFTETECL PCGESEFLDT WNRETHCHQH KYCDPNLGLR VQQKGTSETD TICTCEEGWH CTSEACESCV LHRSCSPGFG VKQIATGVSD TICEPCPVGF FSNVSSAFEK CHPWTSCETK DLVVQQAGTN KTDVVCGPQD RLRALVVIPI IFGILFAILL VLVFIKKVAK KPTNKAPHPK QEPQEINFPD DLPGSNTAAP VQETLHGCQP VTQEDGKESR ISVQERQ (SEQ ID NO: 64).

[0042] As used herein, the term "variable domain" refers to a polypeptide as defined by Kabat et al., Sequences of Immunological Interest, 5 th ed., US Dept. Health & Human Services, Washington, DC (1991). The numbering and positioning of CDR amino acid residues within the variable domain follow the well-known Kabat numbering convention. For example, the Kabat numbering of BMS3h-56-269 (SEQ ID NO: 41) is compared in Table 2 with the same sequence in which amino acids are numbered consecutively. In the Kabat numbering, BMS3h-56-269 has inserted residues 52A, 82A, 82B, and 82C, and is missing residue 100. [Table 2]

[0043] The term "human," when applied to an antibody polypeptide, means that the antibody polypeptide has sequences (e.g., FR and / or CH domains) derived from human immunoglobulin. A sequence is "derived" from a sequence encoding a human immunoglobulin if it is either: (a) isolated from a human individual or from a cell or cell line derived from a human individual; (b) isolated from a library of cloned human antibody gene sequences or human antibody variable domain sequences; or (c) diversified by mutation and selection from one or more of the above polypeptides. An "isolated" compound, as used herein, means that the compound has been removed from at least one component with which it is naturally associated.

[0044] As used herein, "specific binding" refers to binding that exhibits a dissociation constant (K) of about 1 μM or less, as measured, for example, by surface plasmon resonance. d ) refers to the binding of an antigen by an antibody polypeptide with a nucleotide sequence. Suitable assay systems include Biacore (商標) Surface plasmon resonance (SPR) system and Biacore (商標)Kinetic evaluation software (e.g., version 2.1) is included.

[0045] Binding of the antibody polypeptides of the present application to CD40 antagonizes CD40 activity. "CD40 activity" includes, but is not limited to, T cell activation (e.g., induction of T cell proliferation or cytokine secretion), macrophage activation (e.g., induction of reactive oxygen species and nitric oxide in macrophages), and B cell activation (e.g., B cell proliferation, antibody isotype switching, or differentiation into plasma cells). CD40 activity can be mediated by interaction with other molecules. "CD40 activity" includes the functional interaction of CD40 with the following molecules, which are identified by their Uniprot accession numbers in parentheses: CALR (P27797); ERP44 (Q9BS26); FBL (P22087); POLR2H (P52434); RFC5 (P40937); SGK1 (O00141); SLC30A7 (Q8NEW0); SLC39A7 (Q92504); TRAF2 (Q5T1L5); TRAF3 (Q13114); TRAF6 (Q9Y4K3); TXN (Q5T937); UGGT1 (Q9NYU2); and USP15 (Q9Y4E8).

[0046] For example, the "activity" of CD40 includes its interaction with TRAF2. CD40 / TRAF2 interaction activates NF-κB and JNK. See Davies et al., Mol. Cell Biol. 25: 9806-19 (2005). Thus, this CD40 activity can be determined by CD40-dependent activation of cellular NF-κB and JNK compared to a baseline. As used herein, the terms "activate," "activating," and "activated" refer to an increase of at least 10% (e.g., at least 10%, 25%, 50%, 75%, 80%, 90%, 100%, or more) of a given measurable CD40 activity compared to a baseline. CD40 activity is "antagonized" if CD40 activity is reduced by at least 10% (in exemplary embodiments, at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or 100% (i.e., no detectable activity)) compared to the absence of antagonist. For example, an antibody polypeptide may antagonize some or all of CD40 activity without activating CD40. In certain embodiments, the antibody polypeptide does not activate B cell proliferation. In another embodiment, the antibody polypeptide does not activate cytokine secretion by T cells, wherein the cytokine is at least one cytokine selected from the group consisting of IL-2, IL-6, IL-10, IL-13, TNF-α, and IFN-γ.

[0047] The antibody polypeptides of the present disclosure can be administered to human patients while avoiding much of the anti-antibody immune response that is often elicited by the administration of antibodies from other species (e.g., mice). For example, mouse antibodies can be "humanized" by grafting mouse CDRs onto human variable domain FRs according to procedures well known in the art. However, the human antibodies disclosed herein can be produced without the need for genetic manipulation of mouse antibody sequences.

[0048] Anti-CD40 domain antibodies useful in the present disclosure comprise three complementarity-determining regions (CDRs) and four framework regions (FRs), arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDRs contain most of the residues that form specific interactions with the antigen and are primarily responsible for antigen recognition.

[0049] A genus of single variable domain antibody polypeptides that specifically bind to a single CD40 epitope is described in U.S. Application Publication No. 2014 / 0099317, published April 10, 2014, entitled "ANTIBODY POLYPEPTIDES THAT ANTAGONIZE CD40," which is incorporated herein by reference in its entirety. The antibody polypeptides are structurally and functionally characterized, and the data are described in U.S. Application Publication No. 2014 / 0099317, published April 10, 2014. BMS3h-56-269 is an exemplary single variable domain antibody polypeptide that specifically binds to, but does not stimulate, human CD40 disclosed in U.S. Application Publication No. 2014 / 0099317.

[0050] CDRs contain most of the residues that form specific interactions with an antigen. The single variable domains of the antibody polypeptides of the present disclosure comprise CDR1, CDR2, and CDR3 regions that have amino acid sequences identical to or that differ from the CDR1, CDR2, and CDR3 regions of BMS3h-56-269 (SEQ ID NO: 41) by 1, 2, 3, 4, 5, or 6 amino acids, respectively.

[0051] The amino acid sequence of BMS3h-56-269 (SEQ ID NO: 41) is shown below. EVQLLESGGGLVQPGGSLRLSCAASGFTFR DYEMW WVRQAPGKGLERVS AINPQGTRTYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK LPFRFSD RGQGTLVTVSS

[0052] The amino acids of the three complementarity determining regions are underlined. The amino acid sequence of CDR1 is DYEMW (SEQ ID NO: 1). The amino acid sequence of CDR2 is AINPQGTRTYYADSVKG (SEQ ID NO: 2), and the amino acid sequence of CDR3 is LPFRFSD (SEQ ID NO: 3). An exemplary nucleic acid sequence encoding the amino acid sequence of BMS3h-56-269 is as follows: gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgcgtctc tcctgtgcag cctccggatt cacctttcgg gattatgaga tgtggtgggt ccgccaggct ccagggaagg gtctagagcg ggtctcagct attaatccgc agggtacgcg tacatactac gcagactccg tgaagggccg gttcaccatc tcccgcgaca attccaagaa cacgctgtat ctgcaaatga acagcctgcg tgccgaggat accgcggtat attactgtgc gaaacttccg tttaggtttt ccgaccgggg tcagggaacc ctggtcaccg tctcgagc (SEQ ID NO: 72).

[0053] The variable domains of antibody polypeptides provided by the present disclosure comprise CDR1, CDR2, and CDR3 regions that have amino acid sequences identical to those of BMS3h-56-269 (SEQ ID NOS: 1-3, respectively) or that differ by 1, 2, 3, 4, 5, or 6 amino acids from these CDR1, CDR2, and CDR3 regions, respectively. The CDR1 region can vary by up to two amino acids from SEQ ID NO: 1. The CDR2 region can vary by up to three amino acids from SEQ ID NO: 2. The CDR3 region can vary by up to six amino acids from SEQ ID NO: 3. Thus, the variable domain of an antibody polypeptide can comprise: (a) a CDR1 region comprising the amino acid sequence of SEQ ID NO: 1, or a CDR1 region that differs by at most two amino acids from the CDR1 region of SEQ ID NO: 1, (b) a CDR2 region comprising the amino acid sequence of SEQ ID NO: 2, or a CDR2 region that differs by at most three amino acids from the CDR2 region of SEQ ID NO: 2, and (c) a CDR3 region comprising the amino acid sequence of SEQ ID NO: 3, or a CDR3 region that differs by at most six amino acids from the CDR3 region of SEQ ID NO: 3, wherein said single variable domain binds to CD40. The variable domain of an antibody polypeptide can comprise: (a) a CDR1 region consisting of the amino acid sequence of SEQ ID NO: 1, or a CDR1 region that differs by at most two amino acids from the CDR1 region of SEQ ID NO: 1, (b) a CDR2 region consisting of the amino acid sequence of SEQ ID NO: 2, or a CDR2 region that differs by at most three amino acids from the CDR2 region of SEQ ID NO: 2, and (c) a CDR3 region consisting of the amino acid sequence of SEQ ID NO: 3, or a CDR3 region that differs by at most six amino acids from the CDR3 region of SEQ ID NO: 3, wherein said single variable domain binds to CD40.

[0054] Exemplary antibody polypeptides are described in Section 2.5. Further exemplary antibodies are described herein.

[0055] The variable domains of the antibody polypeptides disclosed herein can comprise: (a) a CDR1 region consisting of the sequence X1-Tyr-Glu-Y1-Trp (SEQ ID NO: 4), where X1 is Asp or Gly and Y1 is Met or Leu; (b) a CDR2 region consisting of the sequence Ala-Ile-Asn-Pro-X2-Gly-Y2-Z2-Thr-Tyr-Tyr-Ala-Asp-Ser-Val-A2-Gly (SEQ ID NO: 5), where X2 is Gln, Tyr, His, Trp, or Ala, Y2 is Thr, Asn, Gly, Ser, or Gln, and Z2 is A and (c) a CDR3 region consisting of the sequence X3-Pro-Y3-Z3-A3-B3-C3 (SEQ ID NO: 6), wherein X3 is Leu, Pro, or Glu, Y3 is Phe, Gln, Thr, Met, or Tyr, Z3 is Arg, Tyr, Pro, Leu, Thr, Ile, Phe, Met, or Ser, A3 is Phe or Tyr, B3 is Ser, Gln, His, Asp, Lys, Glu, or Gly, and C3 is Asp, Tyr, Glu, or Ser.

[0056] The variable domains of the antibody polypeptides disclosed herein can comprise: (a) a CDR1 region consisting of the sequence X1-Tyr-Glu-Y1-Trp (SEQ ID NO: 4), where X1 is Asp and Y1 is Met; (b) a CDR2 region consisting of the sequence Ala-Ile-Asn-Pro-X2-Gly-Y2-Z2-Thr-Tyr-Tyr-Ala-Asp-Ser-Val-A2-Gly (SEQ ID NO: 5), where X2 is Gln, Tyr, His, Trp, or Ala and Y2 is Thr, As and (c) a CDR3 region consisting of the sequence X3-Pro-Y3-Z3-A3-B3-C3 (SEQ ID NO: 6), wherein X3 is Leu, Y3 is Phe, Gln, Thr or Met, Z3 is Arg, Tyr, Leu, Thr or Phe, A3 is Phe, B3 is Ser, Gln, His, Asp or Glu, and C3 is Asp or Glu. [Table 3]

[0057] The variable domains of the antibody polypeptides disclosed herein can comprise: (a) a CDR1 region consisting of the amino acid sequence of SEQ ID NO:1; (b) a CDR2 region consisting of the amino acid sequence of SEQ ID NO:2; and (c) a CDR3 region consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22 and SEQ ID NO:23.

[0058] The variable domains of the antibody polypeptides disclosed herein may comprise: (a) a CDR1 region consisting of the amino acid sequence of SEQ ID NO:1; (b) a CDR2 region consisting of the amino acid sequence of SEQ ID NO:27; and (c) a CDR3 region consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26.

[0059] The variable domains of the antibody polypeptides disclosed herein may comprise: (a) a CDR1 region consisting of the amino acid sequence of SEQ ID NO:1; (b) a CDR2 region consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:35 and SEQ ID NO:37; and (c) a CDR3 region consisting of the amino acid sequence of SEQ ID NO:7.

[0060] The variable domains of the antibody polypeptides disclosed herein can comprise: (a) a CDR1 region consisting of the amino acid sequence of SEQ ID NO:1; (b) a CDR2 region consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:36 and SEQ ID NO:38; and (c) a CDR3 region consisting of the amino acid sequence of SEQ ID NO:8.

[0061] The variable domains of the antibody polypeptides disclosed herein may comprise: (a) a CDR1 region consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:39 and SEQ ID NO:40; (b) a CDR2 region consisting of the amino acid sequence of SEQ ID NO:27; and (c) a CDR3 region consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:8 and SEQ ID NO:24.

[0062] The variable domain of an antibody polypeptide disclosed herein may comprise: (a) a CDR1 region consisting of the amino acid sequence of SEQ ID NO: 1; (b) a CDR2 region consisting of the amino acid sequence of SEQ ID NO: 2; and (c) a CDR3 region consisting of the amino acid sequence of SEQ ID NO: 3. The variable domain of an antibody polypeptide disclosed herein may comprise or consist of the amino acid sequence of SEQ ID NO: 41 (the 3h-56-269 sequence).

[0063] The variable domain of an antibody polypeptide disclosed herein may comprise a CDR1 region, a CDR2 region, and a CDR3 region, wherein the amino acid sequence of the CDR1 region, the amino acid sequence of the CDR2 region, and the amino acid sequence of the CDR3 region are selected from the group consisting of: (1) SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively; (2) SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 7, respectively; (3) SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:8, respectively; (4) SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:9, respectively; (5) SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 10, respectively; (6) SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 11, respectively; (7) SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 12, respectively; (8) SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 13, respectively; (9) SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 14, respectively; (10) SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 15, respectively; (11) SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 16, respectively; (12) SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 17, respectively; (13) SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 18, respectively; (14) SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 19, respectively; (15) SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 20, respectively; (16) SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 21, respectively; (17) SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 22, respectively; (18) SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 23, respectively; (19) SEQ ID NO: 1, SEQ ID NO: 27, and SEQ ID NO: 7, respectively; (20) SEQ ID NO: 1, SEQ ID NO: 27, and SEQ ID NO: 8, respectively; (21) SEQ ID NO: 1, SEQ ID NO: 27, and SEQ ID NO: 24, respectively; (22) SEQ ID NO: 1, SEQ ID NO: 27, and SEQ ID NO: 25, respectively; (23) SEQ ID NO: 1, SEQ ID NO: 27, and SEQ ID NO: 26, respectively; (24) SEQ ID NO: 1, SEQ ID NO: 28, and SEQ ID NO: 7, respectively; (25) SEQ ID NO: 1, SEQ ID NO: 29, and SEQ ID NO: 8, respectively; (26) SEQ ID NO: 1, SEQ ID NO: 30, and SEQ ID NO: 7, respectively; (27) SEQ ID NO: 1, SEQ ID NO: 31, and SEQ ID NO: 8, respectively; (28) SEQ ID NO: 1, SEQ ID NO: 32, and SEQ ID NO: 7, respectively; (29) SEQ ID NO: 1, SEQ ID NO: 33, and SEQ ID NO: 8, respectively; (30) SEQ ID NO: 1, SEQ ID NO: 34, and SEQ ID NO: 8, respectively; (31) SEQ ID NO: 1, SEQ ID NO: 35, and SEQ ID NO: 7, respectively; (32) SEQ ID NO: 1, SEQ ID NO: 36, and SEQ ID NO: 8, respectively; (33) SEQ ID NO: 1, SEQ ID NO: 37, and SEQ ID NO: 7, respectively; (34) SEQ ID NO: 1, SEQ ID NO: 38, and SEQ ID NO: 8, respectively; (35) SEQ ID NO: 39, SEQ ID NO: 27, and SEQ ID NO: 8, respectively; (36) SEQ ID NO: 39, SEQ ID NO: 27, and SEQ ID NO: 24, respectively; (37) SEQ ID NO: 40, SEQ ID NO: 27, and SEQ ID NO: 8, respectively; and (38) SEQ ID NO: 40, SEQ ID NO: 27 and SEQ ID NO: 24, respectively.

[0064] The variable domain of the antibody polypeptide can differ from the variable domain of BMS3h-56-269 by up to 10 amino acids, or any integer value therebetween, where the variable domain variant specifically binds to CD40. Alternatively, the variable domain variant can have at least 90% sequence identity to the sequence of BMS3h-56-269 (e.g., at least 92%, 95%, or 98% sequence identity). Non-identical amino acid residues or amino acids that differ between the two sequences can represent amino acid substitutions, additions, or deletions. When the two sequences are aligned using any suitable amino acid sequence alignment algorithm (e.g., BLAST), residues that differ between the two sequences appear as non-identical positions.

[0065] A variable domain can include one or more framework regions (FR) that have the same amino acid sequence as the corresponding framework region encoded by human germline antibody gene segments. For example, a domain antibody can include a V H Germline gene segments DP47, DP45, or DP38, V κ Germline gene segment DPK9, J H Segment JH4b, or J κ Segment J κ It may include 1.

[0066] Exemplary framework regions include those from 3h-56-269: FR1 = EVQLLESGGGLVQPGGSLRLSCAASGFTFR (amino acids 1-30 of 3h-56-269); FR2 = WVRQAPGKGLERVS (amino acids 36-49 of 3h-56-269); FR3 = RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK (amino acids 67-98 of 3h-56-269); FR4 = RGQGTLVTVSS (amino acids 106-116 of 3h-56-269). These sequences correspond to SEQ ID NOs: 42, 44, 47, and 54, respectively, in Table 3. Other exemplary framework regions are shown in Table 3. [Table 4]

[0067] Another exemplary framework region is that of the 3h-56-269 series clone disclosed in U.S. Application Publication No. 2014-0099317.

[0068] Anti-CD40 antibody polypeptides containing mutated IgG1 Fc domains have therapeutic value in the treatment or prevention of immune diseases. Bringing protein therapeutics to market requires molecules with physical and chemical properties suitable for development, commonly referred to as chemistry, manufacturing, and quality control (CMC). The physical and chemical properties of a molecule (including stability, solubility, and homogeneity) are collectively referred to as "developability." Advantageously, anti-CD40 antibody polypeptides containing mutated IgG1 Fc domains exhibit improved developability compared to the same anti-CD40 variable domains linked to other IgF1 and IgF4 Fc domains. Anti-CD40 antibody polypeptides containing mutated IgG1 Fc domains exhibit reduced binding to Fc gamma receptors as measured by SPR and reduced or undetectable iDC activation as measured by at least one of: 1) release of cytokines IL-6 and / or TNF-alpha; and 2) upregulation of cell surface expression of CD86 and / or CD54. Furthermore, the anti-CD40 antibody polypeptides comprising the mutated IgG1 Fc domains have improved thermal stability as measured by DSC and improved physical stability as measured under accelerated stress conditions. The anti-CD40 antibody polypeptides comprising the mutated IgG1 Fc domains have improved homogeneity.

[0069] Linker In some embodiments, the antibody polypeptides of a fusion antibody polypeptide can be linked by an "amino acid linker" or "linker." For example, a dAb can be fused to the N-terminus of the amino acid linker, and an Fc domain can be fused to the C-terminus of the linker. The amino acid linker can be of any length and can consist of any combination of amino acids, although the linker length can be relatively short (e.g., five amino acids or less) to reduce interactions between the linked domains. The amino acid composition of the linker can be adjusted to reduce the number of amino acids with bulky side chains or amino acids that may introduce secondary structure. Suitable amino acid linkers include, but are not limited to, amino acid linkers up to 3, 4, 5, 6, 7, 10, 15, 20, or 25 amino acids in length. The linker AST (SEQ ID NO: 57) can be used in fusion polypeptides. Other exemplary amino acid linker sequences include GGGGS (SEQ ID NO: 58) and linkers containing two, three, four, or five copies of GGGGS (SEQ ID NOs: 59-62, respectively). Table 4 lists exemplary linker sequences used in the present disclosure. [Table 5]

[0070] 2.4. Exemplary Antibody Polypeptides Exemplary antibody polypeptides include: (1) a single variable domain comprising: (a) a CDR1 region comprising the amino acid sequence of SEQ ID NO: 1, or a CDR1 region that differs from the CDR1 region of SEQ ID NO: 1 by at most two amino acids, (b) a CDR2 region comprising the amino acid sequence of SEQ ID NO: 2, or a CDR2 region that differs from the CDR2 region of SEQ ID NO: 2 by at most three amino acids, and (c) a CDR3 region comprising the amino acid sequence of SEQ ID NO: 3, or a CDR3 region that differs from the CDR3 region of SEQ ID NO: 3 by at most six amino acids, wherein the single variable domain binds to CD40; and (2) an Fc domain that is a human IgG1 Fc domain polypeptide comprising a Kabat mutation at position 238 that reduces binding to Fc gamma receptors, wherein the proline at position 238 (P238) is mutated to one of a residue selected from lysine, serine, alanine, arginine, and tryptophan. The single variable domains of the antibody polypeptides described herein antagonize at least one activity of CD40. The antibody polypeptides described herein have improved stability compared to a reference polypeptide having the same single variable domain sequence fused to a wild-type IgG1 Fc domain. Provided are antibody polypeptides comprising: (1) the single variable domain described above, wherein the human IgG1 Fc domain has a substituted lysine at Kabat position 238;

[0071] An exemplary amino acid sequence of a human IgG1 Fc domain polypeptide is as follows: EPKSCDKTHTCPPCPAPELLGG K SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (IgG1a-P238K; SEQ ID NO: 134), EPKSCDKTHTCPPCPAPELLGG KSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (IgG1a-P238K; SEQ ID NO: 66), EPKSCDKTHTCPPCPAPELLGG K SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (IgG1f-P238K; SEQ ID NO: 135), and EPKSCDKTHTCPPCPAPELLGG K SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (IgG1f-P238K; SEQ ID NO: 67).

[0072] Exemplary antibody polypeptides are described above, wherein (a) the CDR1 region consists of the sequence X1-Tyr-Glu-Y1-Trp (SEQ ID NO:4), where X1 is Asp or Gly and Y1 is Met or Leu; (b) the CDR2 region consists of the sequence Ala-Ile-Asn-Pro-X2-Gly-Y2-Z2-Thr-Tyr-Tyr-Ala-Asp-Ser-Val-A2-Gly (SEQ ID NO:5), where X2 is Gln, Tyr, His, Trp, or Ala, Y2 is Thr, Asn, Gly, Ser, or Gln, and Z2 is Arg, L and (c) the CDR3 region consists of the sequence X3-Pro-Y3-Z3-A3-B3-C3 (SEQ ID NO: 6), where X3 is Leu, Pro or Glu, Y3 is Phe, Gln, Thr, Met or Tyr, Z3 is Arg, Tyr, Pro, Leu, Thr, Ile, Phe, Met or Ser, A3 is Phe or Tyr, B3 is Ser, Gln, His, Asp, Lys, Glu or Gly, and C3 is Asp, Tyr, Glu or Ser.

[0073] Exemplary antibody polypeptides are described above, wherein (a) the CDR1 region consists of the amino acid sequence of SEQ ID NO: 1 (CDR1 of 3h-56-269), (b) the CDR2 region consists of the amino acid sequence of SEQ ID NO: 2 (CDR2 of 3h-56-269), and (c) the CDR3 region consists of the amino acid sequence of SEQ ID NO: 3 (CDR3 of 3h-56-269). Further provided is the above-mentioned antibody polypeptide, wherein the amino acid sequence of the single variable domain is set forth in SEQ ID NO: 41 (= the 3h-56-269 sequence).

[0074] Exemplary antibody polypeptides comprise or consist of the following amino acid sequences: EVQLLESGGGLVQPGGSLRLSCAASGFTFRDYEMWWVRQAPGKGLERVSAINPQGTRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKLPFRFSDRGQGTLVTVSS ASTEPKSCDKTHTCPPCPAPELLGG K SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 70).

[0075] Exemplary antibody polypeptides comprise or consist of the following amino acid sequences: EVQLLESGGGLVQPGGSLRLSCAASGFTFRDYEMWWVRQAPGKGLERVSAINPQGTRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKLPFRFSDRGQGTLVTVSS AST EPKSCDKTHTCPPCPAPELLGG K SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 136).

[0076] Exemplary antibody polypeptides comprise or consist of the following amino acid sequences: EVQLLESGGGLVQPGGSLRLSCAASGFTFRDYEMWWVRQAPGKGLERVSAINPQGTRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKLPFRFSDRGQGTLVTVSS AST EPKSCDKTHTCPPCPAPELLGG KSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 71).

[0077] Exemplary antibody polypeptides comprise or consist of the following amino acid sequences: EVQLLESGGGLVQPGGSLRLSCAASGFTFRDYEMWWVRQAPGKGLERVSAINPQGTRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKLPFRFSDRGQGTLVTVSS AST EPKSCDKTHTCPPCPAPELLGG K SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 137).

[0078] 2.5. Antibody Polypeptide Preparation The antibody polypeptides of the present disclosure can be produced and purified in any suitable mammalian host cell line (such as CHO, HEK293, COS and NSO) using only conventional techniques, and then purified using one or a combination of methods (including Protein A affinity chromatography, ion exchange or reverse phase techniques, etc.).

[0079] The present disclosure further provides nucleic acids encoding the antibody polypeptides of the present disclosure. The nucleic acids can be inserted into vectors (e.g., suitable expression vectors such as pHEN-1) (Hoogenboom et al. (1991) Nucleic Acids Res. 19:4133-4137). Further provided are isolated host cells containing vectors and / or nucleic acids encoding the antibody polypeptides of the present disclosure.

[0080] 3. Pharmaceutical Compositions and Methods of Treatment Pharmaceutical compositions comprise a therapeutically effective amount of one or more antibody polypeptides and, optionally, a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include, for example, water, saline, phosphate buffered saline, glucose, glycerol, ethanol, and the like, as well as combinations thereof. Pharmaceutically acceptable carriers may further contain small amounts of auxiliary substances, such as wetting agents, emulsifiers, preservatives, or buffers, which enhance the shelf life or effectiveness of the fusion protein. The composition may be formulated to provide rapid, sustained, or delayed release of the active ingredient after administration. Suitable pharmaceutical compositions and methods for their preparation are well known in the art. See, for example, Remington, The Science and Practice of Pharmacy, A. Gennaro, et al., eds., 21st ed., Mack Publishing Co. (2005).

[0081] The pharmaceutical composition may further comprise an immunosuppressant / immunomodulator and / or an anti-inflammatory substance.

[0082] A method for treating an immune disease in a patient in need of such treatment can include administering a therapeutically effective amount of a pharmaceutical composition to the patient. Antagonizing CD40-mediated T cell activation can inhibit unwanted T cell responses that occur, for example, during autoimmunity, transplant rejection, or allergic reactions. Inhibiting CD40-mediated T cell activation can alleviate the progression and / or severity of these diseases.

[0083] Also provided is the use of an antibody polypeptide of the present disclosure, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for treating an immune disease in a patient in need thereof, which medicament may be administered in combination with, for example, an immunosuppressant / immunomodulator and / or an anti-inflammatory substance.

[0084] As used herein, "patient" refers to an animal (e.g., a mammal, including a human). The patient may have been diagnosed with an immune disorder. "Treatment" or "treat" or "treating" refers to a process that involves alleviating the progression or severity of a symptom, disorder, condition, or disease. "Immune disorder" refers to any disease associated with the development of an immune response (including a cellular immune response and / or a humoral immune response) in an individual. Examples of immune disorders include, but are not limited to, inflammation, allergy, autoimmune disease, or transplant-related disease. "Autoimmune disease" refers to any disease associated with the development of an autoimmune response (including a cellular immune response and / or a humoral immune response) in an individual. An example of an autoimmune disorder is inflammatory bowel disease (IBD), including, but not limited to, ulcerative colitis and Crohn's disease. Other autoimmune disorders include systemic lupus erythematosus, multiple sclerosis, rheumatoid arthritis, diabetes, psoriasis, scleroderma, and atherosclerosis. Transplant-related diseases include graft-versus-host disease (GVHD), acute transplant rejection, and chronic transplant rejection.

[0085] Diseases that may be treated by administering the pharmaceutical compositions of the present disclosure may be selected from the group consisting of Addison's disease, allergies, anaphylaxis, ankylosing spondylitis, asthma, atherosclerosis, atopic allergies, autoimmune diseases of the ear, autoimmune diseases of the eye, autoimmune hepatitis, autoimmune parotitis, bronchial asthma, coronary heart disease, Crohn's disease, diabetes, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barre syndrome, Hashimoto's disease, hemolytic anemia, idiopathic thrombocytopenic purpura, inflammatory bowel disease, immune response to recombinant pharmaceuticals (e.g., factor VII in hemophilia), systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, spondyloarthropathy, thyroiditis, transplant rejection, vasculitis, and ulcerative colitis.

[0086] The pharmaceutical composition can be administered alone or in combination with treatment using immunosuppressants / immunomodulators and / or anti-inflammatory substances (i.e., simultaneously or sequentially). Various immune diseases may require the use of specific adjuvant compounds useful for treating immune diseases, which can be determined on a patient-by-patient basis. For example, the pharmaceutical composition can be administered in combination with one or more suitable adjuvants, such as cytokines (e.g., IL-10 and IL-13) or other immune stimulators (e.g., chemokines, tumor-associated antigens, and peptides). Suitable adjuvants are known in the art.

[0087] Any suitable method or route can be used to administer the antibody polypeptide or pharmaceutical composition. Routes of administration include, for example, oral, intravenous, intraperitoneal, subcutaneous, or intramuscular administration. The therapeutically effective dose of the antibody polypeptide administered will depend on many factors, including, for example, the type and severity of the immune disorder being treated, the use of concomitant therapies, the route of administration of the antibody polypeptide or pharmaceutical composition, and the patient's body weight. A non-limiting range for the therapeutically effective amount of a domain antibody is 0.1 to 20 mg / kg of the patient's body weight, and in some embodiments, 1 to 10 mg / kg.

[0088] 4. Kit Kits useful for treating immune disorders in human patients are provided. In certain embodiments, the kits include (a) a dose of an antibody polypeptide of the present disclosure, and (b) instructional materials for using the antibody polypeptide in a method for treating immune disorders in a human patient as disclosed herein.

[0089] As used herein, the term "instructional material" includes publications, records, drawings, or any other medium of expression that can be used to communicate the usefulness of the compositions and / or compounds of the present invention in a kit. The kit's instructional material can, for example, be attached to a container containing the compounds and / or compositions of the present invention or shipped together with a container containing the compounds and / or compositions. Alternatively, the instructional material can be shipped separately from the container with the intention that the recipient will use the instructional material and the compound cooperatively. Delivery of the instructional material can be, for example, by physical delivery of a publication or other medium of expression that communicates the usefulness of the kit, or can be accomplished by electronic transmission (e.g., computer-based means such as email or download from a website). [Example]

[0090] Materials and Methods: This section describes the materials and methods used in the examples that follow. Additional methods are disclosed in the examples.

[0091] Proteins: Antibody and dAb-Fc proteins were expressed in either HEK293 (a cell line derived from human embryonic kidney cells) or Expi293 cells and purified by standard Protein A affinity chromatography followed by preparative size exclusion chromatography. Some selected samples were expressed and purified from UCOE-CHO cells (samples designated "UCOE-CHO").

[0092] CD40 binding kinetics and affinity: CD40 binding affinity of dAb-Fc and antibody molecules was determined by Biacore by capturing the dAb-Fc or antibody on an immobilized Protein A sensor chip surface and binding human CD40 monomer protein (produced in-house) at 30 microliters per minute (μl / min) in PBS-T pH 7.1 with an association time of 180 seconds and a dissociation time of 360 seconds. (商標)Binding was measured by SPR on a T100 or T200 instrument (GE Healthcare Life Sciences, Marlborough, MA). To characterize binding by avidity, human CD40-Fc (produced in-house) was immobilized on a CM5 sensor chip and dAb-Fc or antibody analytes were probed for binding at 30 μl / min using an association time of 180 s and a dissociation time of 240 s.

[0093] Example 1: Treatment of iDCs with dAb-Fc molecules in the presence or absence of FcγR cross-linking 3h56-269-IgG4.1 is an anti-CD40 dAb-FC(IgG4) fusion protein (SEQ ID NO: 75). As described, for example, in WO2012 / 145673, direct agonist activity of 3h56-269-IgG4.1 has not been observed in B cell or T cell depleted peripheral blood mononuclear cells (PBMC). To further characterize the biological activity and safety profile of 3h56-269-IgG4.1, the effect of 3h56-269-IgG4.1 on immature dendritic cells (iDC) was assayed. The materials and methods used in this example include the following:

[0094] Primary cell isolation and culture: Peripheral blood was collected from normal, healthy human donors. Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized human blood by Ficoll density gradient separation. Monocytes were cultured using the manual EasySep protocol (STEMCELL (商標) Mononuclear cells were isolated from PBMCs according to the protocol described in the International Journal of Clinical Oncology (ICH), published by ICH in 2016. One million isolated monocytes were plated in 6 ml of complete medium (RPMI-1640, 10% heat-inactivated fetal bovine serum, 100 units / ml penicillin-streptomycin; additionally containing IL-4 (100 nanograms / milliliter (ng / ml)) and human GM-CSF (100 ng / ml)) in each well of a 6-well plate and incubated at 37°C and 5% CO for 6 days. The medium was changed every other day and replaced with fresh medium containing the same cytokine concentrations. Immature dendritic cells (iDCs) were collected by centrifugation on day 6, washed extensively, and resuspended in complete medium.

[0095] Immature dendritic cell activation assay: Immature dendritic cells (iDCs) were assayed for activation by assessing the release of specific cytokines and the expression of specific cell surface molecules. Titrations of various biological agents were performed in complete medium and added to replicate 96-well plates. For crosslinking (by addition of CD32a-expressing CHO cells), the antibody being assayed was added to iDCs 30 minutes before the addition of CD32a-expressing CHO cells. The ratio of CD32a-expressing CHO cells to iDCs was 1:6.

[0096] To assess cytokines, cells were incubated at 37°C and 5% CO for approximately 18–20 hours; 150 microliters (μL) of supernatant was removed from each well, diluted 1:5, and assayed for IL-6, TNFα, and IL-12 protein concentrations using commercially available ELISA kits (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions.

[0097] To assess the expression of CD86, ICAM-1 (also known as CD54), and CD83, the remaining cells in the plate from the collected supernatants were pooled into one sample per treatment, transferred to a new 96-well round-bottom (RB) plate, and plated at 4°C. ++ and Mg ++ Wash with D-PBS containing LIVE / DEAD (登録商標) Cells were stained for viability using a Fixable Near-IR Dead Cell Stain Kit (Invitrogen, Carlsbad, CA) for 30 min on ice. ++ and Mg ++ Resuspend in D-PBS, 2% FBS, 0.1% NaN3 (staining buffer) without HCl and add 5 μL / well of human TruStain FcX in staining buffer. (商標)The iDCs were blocked with Fc Receptor Blocking Solution (Biolegend, San Diego, CA). PerCpCy5.5-labeled αCD3, αCD19, and αCD14 (Lin - Immunostaining was performed with BUV395-labeled αCD11c (BD Biosciences, San Diego, CA), APC-labeled αCD86 (Biolegend, San Diego, CA), PE-labeled αCD83 (eBioscience, San Diego, CA), and FITC-labeled αCD54 (Biolegend, San Diego, CA) and incubated at 4°C for 45 minutes. Cells were then washed twice with staining buffer and fixed by adding 100 μl of BD Cytofix Fixation Buffer (BD Bioscience, San Diego, CA) for 15 minutes at room temperature (RT) in the dark. iDCs were then cultured in an LSRII-Fortessa (商標) Flow cytometer (BD Biosciences, San Diego, CA) and FlowJo (登録商標) Expression of CD86, ICAM-1, and CD83 was assessed using analytical software (Tree Star Inc., Ashland, OR).

[0098] CP-870,893 mAb is a well-known agonist CD40 mAb (see, e.g., Vonderheide et al., 2007, J. Clin. Oncol. 25(7): 876-883). In these studies, CP-870,893 mAb (also referred to herein as mAb 134-2141; produced in-house) served as a positive control. A second positive control was a soluble CD40L trimeric molecule (produced in-house) that trimerizes via an isoleucine zipper trimerization motif. In some experiments, CHI-L6 IgG4 (produced in-house), a fusion protein of a non-CD40 binding protein with the IgG4.1 Fc tail, served as a negative control.

[0099] dAb-Fc: The amino acid sequences of the dAb-Fc tested in this experiment are shown in Table 5. In these sequences, the single variable domain residues 3h56-269 are amino acids 1 to 118 (underlined). The linker AST (SEQ ID NO: 57) is double underlined. The unformatted C-terminal residues are the Fc domain. [Table 6]

[0100] Results: The effect of 3h56-269-IgG4.1 on immature dendritic cells (iDCs) was assayed. Upregulation of CD86 and ICAM-1 (CD54) expression, as well as cytokine release (e.g., IL-6, TNF), was assessed. Assays were performed on iDCs from nine different donors. A mild increase in CD86 expression on iDCs was observed with 3h56-269-IgG4.1 at 30 μg / ml in one of nine donors and at 100 μg / ml in two of nine donors, and a similar mild increase in cytokine release was observed in one of nine donors (see Figures 2A–2D). Therefore, it was concluded that the Fc-anti-CD40 dAb fusion 3h56-269-IgG4.1 activated immature dendritic cells (iDCs) in a small subset of donors.

[0101] Immature DCs express both FcgR and CD40 and are sensitive to CD40 activation. Therefore, we investigated the possibility that FcgR-mediated clustering or cross-linking may be involved in the observed iDC activation by 3h56-269-IgG4.1. 3h-56-269-CT (SEQ ID NO: 76) is a fusion of the same anti-CD40 dAb (3h-56-269) with an IgG1 Fc tail with reduced FcgR binding, referred to herein as "CT" or "aba." The CT Fc domain is expressed by orencia. (登録商標)The Fc domain present in 3h-56-269-CT (abatacept, Bristol-Myers Squibb Company, New York, NY) is a fusion of an IgG1 Fc domain modified to reduce Fc domain effector function and remove the interchain disulfide bond in the IgG1 hinge region with the extracellular domain of CTLA-4. 3h-56-269-CT has reduced FcgR binding.

[0102] 3h-59-269-CT was tested at 100 μg / ml using iDCs from nine donors. Compared with a negative control consisting of CHI-L6 IgG4, a fusion protein of a non-CD40 protein with an IgG4 Fc tail, iDCs from the nine donors showed no cytokine release or upregulation of CD86 or CD54. In contrast, 3h56-269-IgG4.1 activated iDCs in a subset of three of the nine donors, with at least one measure of iDC activation observed to be greater than the control. The CD40 agonist mAb 134-214 stimulated CD86 and ICAM expression and cytokine release in all donors tested. See Figure 2E.

[0103] These data suggest a role for the Fc portion of the fusion protein in iDC activation. Specifically, these observations suggest that clustering or cross-linking of FcgR by the IgG4.1 Fc domain of 3h56-269-IgG4.1 on the surface of iDCs may be responsible for the activation observed in a subset of donors. The reduced iDC activation of the 3h-59-269-CT fusion protein is consistent with reduced binding to FcgR receptors, including CD32 (FcgRII) and CD16 (FcgRIII), as assessed by surface plasmon resonance (SPR).

[0104] To further investigate the effect of FcgR-mediated dAb cross-linking on iDC cell surface marker expression and cytokine release, we performed additional experiments in eight blood donors, clustering / cross-linking 3h56-269-IgG4.1 with CHO cells that highly overexpress the low-affinity FcgR CD32a. It should be noted that in these experiments, the CHO cell to iDC ratio was 1:6. This ratio represents an excessive level of clustering / cross-linking, possibly exceeding that expected under normal physiological conditions. Similar to what was observed in the previous iDC study (Figure 2), 3h56-269-IgG4.1, in the absence of cross-linking, induced mild iDC activation, as measured by CD86 and IL-6 production, in a minority of donors compared with the CHI-L6 IgG4 control. In contrast, when incorporated with CD32-overexpressing CHO cells, 3h56-269-IgG4.1 at concentrations of 10 μg / ml or greater resulted in iDC activation similar to that of agonistic CD40 antibody crosslinking, as measured by both cell surface markers and cytokine release (Figure 3).

[0105] Example 2: dAB-Fc molecules with impaired FcgR binding To determine whether further Fc mutations could reduce indirect iDC activation mediated by FcgR clustering or cross-linking, other dAb-Fc molecules were produced with mutations in the Fc domain to reduce FcgR binding. FcgR binding affinity was characterized by SPR. The materials and methods used in this example include the following.

[0106] FcgR binding SPR: FcgR binding was measured using purified FcgR and Biacore. (商標) It can be measured in vitro using surface plasmon resonance (SPR). Two methods were used here.

[0107] One method tests the binding of purified antibody or dAb-Fc proteins to His-tagged FcgR proteins (FcgR-His) captured on immobilized Fab fragments of anti-His antibodies. These experiments were performed by Biacore. (商標) T100 or Biacore (商標) Tests are performed at 25°C on either a T200 instrument (GE Healthcare). Fab fragments from a mouse anti-6xHis antibody (produced in-house) are immobilized onto a CM5 sensor chip at a density of approximately 3000 resonance units (RU) in a running buffer of 10 millimolar (mM) HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant p20 (HBS-EP+) using standard ethyl(dimethylaminopropyl)carbodiimide (EDC) / N-hydroxysuccinimide (NHS) chemistry with ethanolamine blocking. All remaining tests are performed using a running buffer of 10 mM NaPO4, 130 mM NaCl, 0.05% surfactant p20 (PBS-T), pH 7.1. Various FcgR proteins (produced in-house) containing a C-terminal 6x polyhistidine tag were captured onto this surface at 10 μl / min for a contact time of 30 seconds (s) (usually using an FcgR-His protein concentration of approximately 7 μg / ml). Various concentrations of purified antibody or dAb-Fc protein are examined for binding (e.g., using an association time of 120 seconds at 30 μl / min and a dissociation time of 120 seconds at 30 μl / min). The FcgR proteins tested in these studies include the "high affinity" FcgR hCD64 (hFcgRI), and the "low affinity" FcgR hCD32a-H131 (FcgRIIa-H131), hCD32a-R131 (FcgRIIa-R131), hCD32b (FcgRIIb), hCD16a-V158 (FcgRIIIa-V158), hCD16a-F158 (FcgRIIIa-F158), hCD16b-NA1 (FcgRIIIb-NA1), and hCD16b-NA2 (FcgRIIIb-NA2).

[0108] To quantitatively analyze the binding response and compare FcgR binding of various molecules, SPR binding data can be analyzed by calculating the maximum binding response as a percentage of the theoretically maximum binding response (%Rmax), generally as shown in Equation 1:

number

number

[0109] "%Rmax analysis" is particularly useful for assessing the binding of "low affinity" FcgRs (e.g., hCD32a-H131, hCD32a-R131, hCD32b, hCD16a-V158, hCD16a-F158, hCD16b-NA1, and hCD16b-NA2, which have relatively fast binding and dissociation rates and affinities near or below the analyte concentration tested (1 micromolar (μM)), such that surface saturation is generally not achieved under these conditions). In contrast, the "high affinity" FcgR hCD64 binds with higher affinity and slower dissociation kinetics than other FcgRs (particularly IgG1 and IgG4), such that these isotypes typically saturate the hCD64 surface at micromolar analyte concentrations, making it more difficult to distinguish affinities using %Rmax. For these interactions, differences between antibodies can be easily observed by comparing dissociation rates in sensorgram data.

[0110] A second SPR assay to investigate the interaction between antibody or dAb-Fc proteins and FcgR proteins is the Protein A capture method. These experiments were performed by Biacore. (商標) T100 or Biacore (商標)These studies are performed at 25°C on either a T200 instrument (GE Healthcare). For these studies, Protein A is immobilized onto flow cells 1-4 of a CM5 sensor chip at a density of approximately 3000 RU in a running buffer of 10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.05% surfactant p20 using standard ethyl(dimethylaminopropyl)carbodiimide (EDC) / N-hydroxysuccinimide (NHS) chemistry with ethanolamine blocking. Antibody or dAb-Fc proteins (typically approximately 3-10 μg / ml) are captured onto the Protein A surface, and FcgR analyte binding is examined (e.g., using a 120-second association time and a 180-second dissociation time at a flow rate of 30 μL / min) in a running buffer consisting of 10 mM NaPO, 130 mM NaCl, 0.05% surfactant p20, and buffer (PBS-T), pH 7.1, at 25°C.

[0111] Protein A capture assays can be used to analyze crude supernatants containing antibody or dAb-Fc molecules. For this analysis, antibody or dAb-Fc proteins can be captured from either undiluted supernatants or supernatants diluted with running buffer. To quantitatively analyze binding responses and compare FcgR binding of various molecules, SPR binding data can be analyzed by calculating %Rmax using Equation 1 above, where the analyte is purified FcgR protein and the ligand is captured antibody or dAb-Fc protein.

[0112] In addition to %Rmax analysis, quantitative analysis of binding kinetics and affinity can be performed by examining titrations of FcgR analytes for binding to Protein A-captured antibodies or dAb-Fc proteins. For example, FcgRs in 3:1 serial dilutions can be titrated from 10 μM to either 0.15 nM (hCD64) or 1.5 nM (all other FcgRs). These kinetic data can be analyzed by Biacore. (商標) The T200 evaluation software can be used to fit a 1:1 Langmuir model or a steady-state binding model to obtain kinetic and affinity values.

[0113] dAb-Fc: The dAb-Fc tested in this example include the dAb-Fc shown in Table 5. The amino acid sequences of additional dAb-Fc tested in this experiment are shown in Table 6. In these sequences, the single variable domain 3h56-269 residues are amino acids 1-118 (underlined). The linker AST (SEQ ID NO: 57) is double underlined. The C-terminal residues are the Fc domain. [Table 7]

[0114] Control mAb: A control monoclonal antibody (1F4) was formatted with a similar Fc domain variant. The antibody does not bind to CD40. SEQ ID NO: 80 in Table 7 is the sequence of the control antibody heavy chain variable region (underlined) and CH1, and SEQ ID NO: 81 is the sequence of the light chain variable region (underlined) and CL. The variously formatted heavy chains are shown in Table 7 as SEQ ID NOs: 82-87. The sequences of the IF4 heavy chain variable region and CH1 region are underlined in SEQ ID NOs: 82-87. The heavy and light chain sequence pairs for each 1F4 mAb variant are shown in Table 8. [Table 8-1] [Table 8-2] [Table 9]

[0115] Results: We generated dAb-Fc molecules with mutations in the Fc domain that reduce FcgR binding. Specifically, we constructed the anti-CD40 domain antibody 3h56-269 with the following Fc domain variants: IgG1.1f, IgG1.3f, and IgG1-D265A. In 3h-56-269-IgG1.1f (SEQ ID NO: 77), 3h-56-269-IgG1.3f (SEQ ID NO: 78), and 3h-56-269-IgG1-D265A (SEQ ID NO: 79), amino acids 1 to 116 represent the 3h-56-269 dAb, amino acids 117 to 119 represent a linker, and amino acids 120 to 351 represent the Fc domain.

[0116] Each of these dAb-Fc fusion proteins, as well as 3h56-269-IgG4.1 and 3h56-269-CT, were analyzed by Biacore. (商標) High affinity binding to purified human CD40 monomer (hCD40 monomer, produced in-house) was confirmed as measured by SPR. As shown in Table 9, the KD values ​​for the various Fc variants range from 7.3 nM to 11.5 nM. As measured by SPR using hCD40-Fc on the surface of a sensor chip and dAb-Fc molecules as soluble analytes in solution, each dAb-Fc molecule binds to human CD40 with high avidity, where data for injections of 250 nM and 25 nM dAb-Fc analyte were fitted to a 1:1 Langmuir model, and the apparent KD values ​​(KD) were influenced by the avidity of all dAb-Fc. apparent ) was estimated to be less than 1 nM. See Table 9. [Table 10] *3h-56-269-CT expressed and purified from UCOE-CHO cells.

[0117] The FcgR binding properties of dAb-Fc molecules and various control monoclonal 1F4 antibodies were characterized by SPR. Initial assays involved binding of 1 μM or 10 μM dAb-Fc or a human IgG1f antibody control (1F4-IgG1f) to an FcgR-His surface captured by anti-His Fab. These data are shown in Table 10. [Table 11]

[0118] In separate assays, FcgR analytes (1 μM or 10 μM) were tested for binding to Protein A-captured dAb-Fc surfaces (data shown in Table 11) and antibody surfaces (data shown in Table 12). [Table 12] [Table 13]

[0119] Based on the binding response, or lack thereof, in these experiments, a subset of higher affinity dAb-Fc / FcgR or Ab / FcgR interactions with the strongest binding responses were selected for kinetic / affinity characterization using analyte titration (binding of FcgR analyte to antibody or dAb-Fc captured on Protein A). These data are shown in Table 13. [Table 14]

[0120] Taken together, these FcgR binding SPR data indicate that IgG1f and IgG4.1 isotype molecules have significantly higher FcgR affinity at all FcgRs compared to the modified Fc variants IgG1-D265A, IgG1.1f, IgG1.3f, or CT molecules. Among the modified Fc variants, hCD64 binding affinity was strongest for 3h56-269-CT (KD = 4.6 nM), weaker for 3h56-269-IgG1-D265A (KD = 62 nM), and weakest for 3h56-269-IgG1.1f and 3h56-269-IgG1.3f, which was too weak to be quantified under the conditions tested (KD = 4.6 nM). D >5 μM, which is half of the highest analyte concentration tested). All other FcgR interactions of IgG1-D265A, IgG1.1f, IgG1.3f, and CT variants (hCD32a-H131, hCD32a-R131, hCD32b, hCD16a-V158, hCD16b-NA2) were too weak to yield reliable KD values ​​(K D >5 μM). However, differences in relative binding responses can be observed in the %Rmax data. For example, the IgG1-D265A variant has a stronger binding response for hCD32a-H131 compared to the IgG1.1f, IgG1.3f, or CT variants (Table 11). In contrast, the IgG1.1f and IgG1.3f variants have a stronger binding response for hCD32a-R131 compared to the IgG1-D265A and CT variants (Table 11).

[0121] The dAb-Fc molecules were tested in an iDC assay (described in Example 1) with and without crosslinking of CD32-overexpressing CHO cells. These data are shown in Figure 4. The 3h-59-269-CT molecule did not result in iDC activation above control levels at concentrations up to 100 μg / ml (left panel of Figure 4), even when crosslinked with CD32-expressing CHO cells (right panel of Figure 4). However, domain antibody Fc fusions with changes designed to minimize FcgR binding (3h-59-269-IgG1.1f and 3h-59-269-IgG1.3f) showed reduced (but still measurable) iDC activation, as measured by upregulation of CD54 (also known as ICAM1) and CD86 expression and increased cytokine release from at least one of the four donors tested at the highest concentration of 100 μg / ml. The inclusion of CD32-expressing CHO cells to induce cross-linking resulted in robust iDC activation, as measured by upregulation of CD86 and CD54 expression in all four donors. These data demonstrate the FcgR dependence of the observed iDC activation.

[0122] Example 3: Evaluation of the developability of dAb-Fc proteins Bringing protein therapeutics to market requires molecules with suitable physical and chemical properties for development, commonly referred to as chemistry, manufacturing, and control (CMC). A molecule's physical and chemical properties (including stability, solubility, and homogeneity) are collectively referred to as "developability." Many techniques and assays have been developed to evaluate the developability of candidate protein therapeutic molecules, including differential scanning calorimetry (DSC), imaging capillary isoelectric focusing (icIEF), mass spectrometry (MS), and accelerated stability studies.

[0123] The developability of various dAb-Fc proteins was evaluated by DSC, icIEF and mass spectrometry. Materials and methods are described below.

[0124] Differential Scanning Calorimetry: DSC experiments were performed on a MicroCal VP-Capillary DSC instrument (Malvern Instruments, Malvern, UK) in 10 mM NaPO, 130 mM NaCl pH 7.1. 1 mg / ml dAb-Fc or antibody samples were tested using a scan range of 10-110 °C and a scan rate of 90 °C / hr. Data were analyzed using MicroCal-Origin 7.0 software.

[0125] Imaged capillary isoelectric focusing: icIEF experiments performed with ProteinSimple iCE3 (商標) For these studies, dAb-Fc or antibody samples, typically at a concentration of 2 mg / ml, were mixed with a carrier ampholyte mixture consisting of 2 M urea, 0.35% methylcellulose, 1% Pharmalyte 5-8, 3% Pharmalyte 8-10.5, and pI markers 5.85 and 10.10 to a final protein concentration of 0.20 mg / ml and analyzed at 1.5 kV with a 1-minute pre-focusing time and 3 kV with a 10-minute focusing time.

[0126] Mass spectrometry: For mass spectrometry (mass spec), samples were reduced with 100 mM DTT and N-deglycosylated with peptide:N-glycosidase (FPNGaseF). The liquid chromatography-mass spectrometry (LC / MS) system used was a Waters Acquity (登録商標) Waters Synapt with UPLC (Ultra High Performance Liquid Chromatography) (登録商標) The UPLC column was a Waters Acquity G2 (Waters Corporation, Milford, MA). (登録商標)The column was BEH (ethylene bridged hybrid particles) C4 (2.1x150mm, 300Å, 1.7um particles). The gradient was 10% to 38% (mobile phase B) in 10 minutes at a flow rate of 200μL / min. Mobile phase A was 0.1% formic acid in water. Mobile phase B was 0.1% formic acid in acetonitrile. The column temperature was 60°C. Data analysis was performed using Waters MassLynx. (商標) The software was used to perform the analysis manually; deconvolution of the spectra was performed using the MaxEnt1 algorithm.

[0127] Accelerated Stability Studies: Accelerated stability studies were performed by first extensively dialysis of the dAb-Fc molecules into the target formulation buffer at 4°C. Samples were collected and analyzed by Amicon (登録商標) The samples were concentrated using ultra centrifugal filter units (Merck KgaA, Germany) and adjusted to various target concentrations with dialysis buffer. These samples were incubated for several weeks at various temperatures (usually 4°C, 25°C, 32°C, and / or 40°C) while aliquots were removed and analyzed by analytical size exclusion chromatography. Analytical size exclusion chromatography was performed using a Shodex (商標) The analysis was performed on an Agilent 1260 HPLC using a K403-4F column (Showa Denko America, Inc., New York, NY) with a mobile phase of 100 mM sodium phosphate, 150 mM sodium chloride, pH 7.3 at a flow rate of 0.3 ml / min.

[0128] Results - Differential Scanning Calorimetry: DSC can be used to measure the thermal stability of proteins. DSC data for 3h56-269 dAb constructed with various Fc domains is shown in Figure 5. Optimal Tm values ​​are summarized in Table 14. [Table 15]

[0129] Based on the characteristic thermal denaturation profile of the IgG Fc domain, the transition of the Fc CH3 domain of 3h56-269-IgG4.1 was assigned as a transition with a midpoint (Tm) value of 69.6 °C; the Fc CH3 domains of various IgG1 molecules were assigned as transitions with Tm of approximately 82–83 °C. The denaturation of the dAb domain and CH2 domain of dAb-Fc was assigned to a transition below 65 °C, which corresponds to the onset of thermal denaturation (T onset ), the shape of the unfolding transition and the optimal Tm values ​​all differed among the various constructs. For example, the thermal transitions of the dAb and CH2 domains of 3h56-269-IgG4.1 occur as a single overlapping transition or cooperative transition with a Tm value of 62.8 °C. The unfolding profiles of the dAb and CH2 domains of 3h56-269-IgG1-D265A, 3h56-269-IgG1.1f, and 3h56-269-IgG1.3f all corresponded to more asymmetric transitions, which were best described by two transitions with Tm values ​​of approximately 56–63 °C. 3h56-269-CT had the lowest T onset It had a broad thermal transition and the lowest compatible Tm values ​​of Tm1=55.4°C and Tm2=60.4°C, with an onset of unfolding at approximately 40°C.

[0130] Results - Imaging Capillary Isoelectric Focusing (icIEF): Imaging capillary isoelectric focusing (icIEF) can be used to characterize the homogeneity or heterogeneity of a sample. The ability to generate a homogeneous product is another important developability criterion. As a result, during the discovery and optimization of novel protein therapeutics, various analytical methods are utilized to characterize and quantify sample heterogeneity and select the most homogeneous molecules.

[0131] The charge profiles of the dAb-Fc molecules were characterized by icIEF. The data are shown in Figure 6. The icIEF profiles of 3h56-269-IgG4.1 (Figure 6A), 3h56-269-IgG1.1f (Figure 6E), and 3h56-269-IgG1.3f (Figure 6F) were all relatively simple, with each profile consisting of a clear major peak with an area of ​​69–86% and two to four lower-abundance charge variants. This icIEF profile resembles a typical profile obtained for antibodies. The major peak of 3h56-269-IgG1-D265A (Figure 6D) is slightly lower in abundance (49%), with corresponding higher levels of acidic variants, including at least six detectable species. In contrast, the profile of 3h56-269-CT (Figure 6B) is highly heterogeneous, consisting of at least 16 species and lacking a clear major peak. The icIEF profile of 3h56-269-CT expressed in a different cell line (UCOE-CHO) was similarly heterogeneous (Fig. 6C), although the distribution of charge variants was significantly different from that expressed in HEK293.

[0132] Results—Mass Spectrometry: Typical glycosylation on the Fc domain of IgG or Fc-containing proteins is a mixture of G0F, G1F, and some G2F species. Other glycoforms (such as sialylated or non-fucosylated) are generally found at very low abundance or undetectable levels.

[0133] Mass spectrometry experiments were performed to characterize the glycosylation profile of the dAb-Fc proteins and compare them to a control antibody with similar Fc mutations. The data are shown in Table 15. [Table 16]

[0134] Mass spectrometry data for the control antibodies 1F4-IgG1f and 1F4-IgG1.3f, and the dAb-Fc antibodies 3h56-269-IgG4.1, 3h56-269-IgG1.1f, and 3h56-269-IgG1.3f, showed that these proteins consisted of a typical mixture of G0F and G1F glycoforms, with a lower abundance of the G2F species.

[0135] Both dAb-Fc and antibody molecules containing the D265A mutation in the Fc domain contained a mixture of G0F, G1F, and G2F species, but also had higher levels of sialylated glycoforms. All of these D265A molecules could be deglycosylated using standard PNGase enzyme treatment protocols; this data is consistent with the glycans of the D265A molecules being N-linked and occupying the common Asn297 residue in the Fc domain.

[0136] In contrast, mass spectrometry data for 3h56-269-CT or the control 1F4-CT antibody expressed in either HEK293 or UCOE-CHO cells revealed that these proteins were highly heterogeneous, with evidence of many different multiply glycosylated species, including highly sialylated species. The data for 3h56-269-CT are shown in Table 16. [Table 17-1] [Table 17-2]

[0137] Mass spectrometry data for the control 1F4-CT antibody is shown in Table 17. [Table 18]

[0138] Furthermore, the 3h56-269-CT and 1F4-CT molecules could not be effectively deglycosylated by treatment with PNGase; these results suggest that at least some of the complex glycans were O-linked at Ser or Thr residues. These data are consistent with the known glycosylation of abatacept, which contains the same modified IgG1 Fc domain (containing the C220S, C226S, C229S, and P238S mutations). In abatacept, these introduced Ser mutations have been shown to represent O-linked glycosylation sites in the hinge region, which is heterogeneously glycosylated and enriched for sialic acid species.

[0139] Results—Accelerated Stability Studies: Because 3h56-269-CT was the only dAb-Fc molecule that showed no response in the iDC assay, either in the absence or presence of crosslinking of CD32-overexpressing CHO cells, it was selected for further studies, including evaluation of its potential for further development. Specifically, stability studies were performed under accelerated stress conditions at low temperatures of 32°C and 40°C, as well as 4°C and 25°C. The formulation buffer for these studies (20 mM potassium phosphate, 250 mM sucrose, 50 μM DTPA, and 0.05% PS80, pH 7.0) was selected based on screening the molecule's thermal stability using the UNit platform (Unchained Labs, Woburn, MA) to identify conditions that confer favorable thermal stability (Tm) and onset of aggregation (Tag). Purified 3h56-269-CT protein was exchanged into this formulation buffer by dialysis, concentrated, and adjusted to a final concentration of 50 mg / ml or 150 mg / ml and incubated at various temperatures for 4 weeks. To assess the physical stability of the protein, aliquots were removed at time zero (t0), 1 week (1w), and 4 weeks (4w) after the start of incubation at various temperatures. Samples were analyzed by analytical size exclusion chromatography (aSEC) to determine the levels of monomeric protein, high molecular weight aggregates (HMW), and low molecular weight species (LMW). HMW data are shown in Table 18. [Table 19]

[0140] The aSEC data showed high levels of HMW formation of 3h56-269-CT, especially at higher protein concentrations and higher temperatures.

[0141] Example 4: Fc domain variants As shown in Examples 1 and 2, the 3h56-269-CT molecule was found to have advantageously weak FcgR binding (especially to low-affinity FcgRs (hCD32a, hCD32b, hCD16a, and hCD16b)) and demonstrated a lack of response in iDC assays involving crosslinking of CD32-overexpressing CHO cells. However, as shown in Example 3, biophysical characterization of 3h56-269-CT indicated that the molecule had low thermal stability, high heterogeneity, and poor physical stability. Therefore, efforts were initiated to improve the 3h56-269-CT molecule while maintaining its advantageously weak FcgR binding and lack of signal in iDC assays by reducing or eliminating O-linked glycans, reducing or eliminating sialic acid content, reducing heterogeneity, and improving thermal and physical stability.

[0142] To attempt to improve the biophysical characteristics of 3h56-269-CT, a series of mutant dAb-Fc molecules were designed to understand the contribution of the individual C220S, C226S, C229S, and P238S mutations to the properties of 3h56-269-CT, and to separate the undesirable developability issues from the desirable weak FcgR binding and lack of Fc-mediated signaling. The mutation strategy involved the design of several variants at positions 220, 226, 229, and 238 (Kabat numbering). The following variants were designed:

[0143] a) A set of single and combined Ser mutants at positions 220, 226, 229 and 238 to test the individual and combined effects of these mutations. See SEQ ID NOs: 88-96 in Table 19. The underlined sequences are anti-CD40 single variable domains. [Table 20-1] [Table 20-2]

[0144] b) A set of single and combined Ala mutants or combined Ala and Ser mutants at positions 226, 229, and 238 to identify the primary site of O-linked glycosylation and its effect on molecular properties. Similar to the Ser mutations, the Ala mutations at C220, C226, and C229 are predicted to prevent disulfide bond formation. However, unlike Ser, the Ala residues are not sites of O-linked glycosylation. See SEQ ID NOs: 97-109 in Table 20. [Table 21-1] [Table 21-2] [Table 21-3]

[0145] c) A set of mutants with P238 mutated to lysine (P238K) was designed to test whether a non-conserved positively charged residue at this position in the downstream hinge region could reduce FcgR binding affinity (see SEQ ID NOs: 110-116 in Table 21). [Table 22-1] [Table 22-2]

[0146] d) For both the IgG1a and IgG1f allotypes, dAb-Fc molecules were generated containing the L234A, L235A mutation (abbreviated as "LALA"). See SEQ ID NOs: 117-118 in Table 22. [Table 23]

[0147] e) dAb-Fc molecules containing a single N297A mutation were generated for both the IgG1a and IgG1f allotypes. See SEQ ID NOs: 119-120 in Table 23. [Table 24]

[0148] In addition to the dAb-Fc variants, a smaller set of related Fc mutants was designed to determine whether similar mutations on the full IgG would have a similar effect on properties as the dAb-Fc configuration. All IgG variants were generated using the variable domain of the control 1F4 antibody. The heavy chain sequences of these variants are shown in Table 24. The sequence of the portion of the 1F4 heavy chain containing the variable and CH1 regions (SEQ ID NO: 80) is shown in italics. For each of these variant 1F4 monoclonal antibodies, the light chain sequence was SEQ ID NO: 81 (see Table 7). The variants included the following:

[0149] a) Single and double C226S and C229S variants, see SEQ ID NOs: 121-123 in Table 24.

[0150] b) Single and double C226A and C229A variants, see SEQ ID NOs: 124-126 in Table 24.

[0151] c) P238S and P238K variants, see SEQ ID NOs: 127-128 in Table 24.

[0152] d) The C226S, C229S, P238S triple mutant was tested for the IgG1f allotype, see SEQ ID NO: 129 in Table 24.

[0153] e) The N297A mutation was tested on the IgG1f allotype. See SEQ ID NO: 130 in Table 24. [Table 25-1] [Table 25-2] [Table 25-3]

[0154] The heavy and light chain sequence pairs for each 1F4 mAb variant are shown in Table 25. [Table 26]

[0155] All 1F4-IgG variants were generated with the wild-type Cys220 residue unchanged to pair with the C-terminal Cys residue of the antibody light chain.

[0156] Example 5: Characterization of 1F4 control antibodies containing variant Fc domains To characterize the FcgR binding properties of the Fc-engineered 1F4 antibody molecule, SPR experiments were performed by testing the binding of 1 μM or 10 μM purified antibody analyte to anti-His-captured FcgR surfaces as described in Example 2. Binding responses were analyzed and presented as %R values; the results are shown in Table 26. [Table 27]

[0157] These data indicate that a single Cys-to-Ser mutation at position 226 (1F4-IgG1a-C226S) or 229 (1F4-IgG1a-C229S) in the hinge region has only a minor effect on FcgR binding compared to the wild-type IgG1f antibody (1F4-IgG1f). The C226S, C229S double mutant (1F4-IgG1a-C226S-C229S) has significantly weaker binding to all low-affinity FcgR proteins; however, this binding response is significantly stronger than that of the 1F4-CT molecule. These data suggest that the additional P238S mutation in the 1F4-CT molecule further contributes to the reduced FcgR binding.

[0158] The single C226A (1F4-IgG1a-C226A) or C229A (1F4-IgG1a-C229A) mutants bound to FcgR similarly to the single C226S or C229S mutants; similarly, the C226A, C229A double mutant (1F4-IgG1a-C226A-C229A) bound to FcgR similarly to the C226S, C229S double mutant (1F4-IgG1a-C226S-C229S). Ala mutations at these sites prevent inter-heavy chain disulfide bond formation, similar to Ser mutations at these sites. However, unlike Ser mutations, Ala mutations are not O-glycosylation sites. Therefore, these data suggest that O-glycosylation at S226 and / or S229 does not significantly affect FcgR binding.

[0159] The P238K and N297A variants (1F4-IgG1a-P238K and 1F4-N297A, respectively) showed the weakest binding responses to low-affinity FcgRs, showing essentially no detectable binding signals to hCD32a-H131, hCD32a-R131, hCD32b, hCD16a-V158, or hCD16b-NA2. The 1F4-IgG1a-P238K variant showed weaker FcgR binding than the 1F4-IgG1a-P238S variant, suggesting that Lys at position 238 is more effective at disrupting FcgR binding than Ser at that position. Furthermore, the SPR sensorgram data showed that the dissociation rates of 1F4-IgG1f-N297A and 1F4-IgG1a-P238K binding to hCD64 were significantly faster than those of 1F4-IgG1f or 1F4-CT (see Figure 7).

[0160] The thermal stability of the Fc variant 1F4 antibody was characterized by DSC as described in Example 3. Thermal transitions were assigned to either the CH2 domain, CH3 domain, or Fab domain based on the well-characterized thermal denaturation profile of the IgG molecule. The optimal Tm values ​​are summarized in Table 27. [Table 28]

[0161] The Fab domain of the 1F4 antibody has an appropriate Tm of 71.6°C to 74.7°C. The CH3 domains of all molecules melted at 82.1°C to 83.1°C, which is typical of wild-type (unmodified) IgG1 CH3 domains. The CH2 domain is the least stable domain of the antibody, and the melting temperatures varied among the various mutants, suggesting that mutations in the hinge / CH2 region affect the thermal stability of the CH2. The Tm values ​​of the CH2 domains of 1F4-CTf (54.3°C) and 1F4-CT (55.1°C) differed by less than 1°C, suggesting that the IgG1 allotype only slightly affects the thermal stability of the CH2 domain. However, these CH2 domains were significantly destabilized (approximately 17-18°C) compared to the wild-type CH2 domain of 1F4-IgG1f (72.2°C). These data are consistent with the low thermal stability observed for the CH2 / dAb domain of 3h56-269-CT.

[0162] Fc mutants with a single Cys-to-Ser mutation in the hinge region slightly reduced the stability of the CH2 domain compared to wild-type IgG1f, with the Tm values ​​of the CH2 domain being 70.3°C for 1F4-IgG1a-C226S and 69.9°C for 1F4-IgG1a-C229S. Mutation of both hinge Cys residues to Ser further reduced the Tm of the CH2 domain of 1F4-IgG1a-C226S and C229S to 64.8°C. The single P238S mutation reduced the stability of the CH2 domain (62.4°C) compared to wild-type 1F4-IgG1f. Therefore, these data indicate that the combination of all three mutations (C226S, C229S, and P238S) in 1F4-CT significantly destabilizes the CH2 domain, rather than that any of the three individual mutations alone are responsible for the reduced stability of the CH2 domain.

[0163] The single Cys→Ala mutants of the hinge 1F4-IgG1a-C226A and 1F4-IgG1a-C229A have CH2 domain Tm values ​​almost identical to those of the Cys→Ser mutants at these positions, whereas the double mutant 1F4-C226A,C229A has a CH2 domain Tm that is slightly (1.2°C) more stable than that of the Cys→Ser double mutant 1F4-C226S,C229S. The CH2 domain of 1F4-IgG1a-P238K (Tm = 64.0°C) is 1.6°C more stable than the Ser mutant 1F4-IgG1a-P238S (Tm = 62.4°C) at this position.

[0164] To determine the effect of the hinge / Fc mutations on sample heterogeneity, the 1F4-IgG molecule was characterized by icIEF as described in Example 3. The icIEF profile of the 1F4-IgG1f protein was typical of a monoclonal IgG1 antibody, with a major peak at 79.7% abundance and approximately two to four acidic or basic variants at much lower abundance (see Figure 8). Similar to the domain antibody with the CT Fc domain (3h56-269-CT), the icIEF profile of the 1F4-CT molecule was heterogeneous, consisting of at least eight different charge variants, with no clearly dominant species. This heterogeneity may be related to the glycan heterogeneity observed by mass spectrometry (Table 17), as described above.

[0165] The icIEF data for the double Cys→Ser variant 1F4-IgG1a-C226S, C229S were similar to those for the 1F4-CT molecule, showing the presence of numerous differently charged variants without a clear major peak. The data for the single mutants C226S, C229S, and P238S all showed a complexity similar to that of 1F4-IgG1f. These data suggest that both the C226S and C229S mutations are required for high levels of O-linked sialic glycosylation in the hinge / Fc region, and that these mutations together disrupt the inter-heavy chain hinge disulfide bond.

[0166] The icIEF data for 1F4-IgG1.3f, 1F4-N297A, 1F4-IgG1a-P238K and their respective single and double Ala mutants showed similar homogeneity to that of 1F4-IgG1f, each consisting of a major peak of 62–80% abundance with minor amounts of approximately two to three acidic or basic variants.

[0167] Taken together, the icIEF data show that all molecules in which both the hinge Cys226 and Cys229 residues are mutated to Ser have significantly higher heterogeneity than the other variants.

[0168] Control antibody data summary: SPR, DSC, icIEF and mass spectrometry data of the 1F4-IgG molecule provide insight into the role of the C226, C229 and P238 mutations on FcgR binding, thermal stability and heterogeneity of the CT Fc domain.

[0169] The single hinge C226S and C229S mutants exhibited slightly reduced thermal stability and heterogeneity and FcgR binding similar to 1F4-IgG1f, whereas the C226S, C229S hinge double mutant exhibited significantly reduced thermal stability, increased heterogeneity, and reduced FcgR binding compared to 1F4-IgG1f. The single P238S mutation had a similar effect on reduced thermal stability and FcgR binding as the C226S, C229S double mutant, but did not increase heterogeneity. Combining the C226S, C229S hinge mutation with P238S to produce the complete 1F4-CT molecule exhibited heterogeneity similar to the single C226S, C229S mutants, but further reduced thermal stability and FcgR binding. Taken together, these data suggest that the combination of the C226S and C229S mutations plus P238S contributes to reduced thermostability and FcgR binding compared to wild-type Fc, respectively, and suggest that the primary sites of O-linked glycosylation are on the mutant hinge residues S226 and Ser229.

[0170] Single and double Cys-to-Ala mutations at positions 226 and 229 in the hinge region exhibit similar thermostability and FcR binding to Cys-to-Ser mutants at those sites. However, the C226A and C229A mutants lack the O-linked glycosylation site at the Ser residue and do not exhibit the high heterogeneity observed with the C226S and C229S mutants. This suggests that O-linked glycosylation in the hinge region does not significantly affect FcR binding.

[0171] The 1F4-IgG1a-P238K mutant exhibited weaker FcgR binding than 1F4-IgG1a-P238S, but had similar heterogeneity and superior thermal stability compared to 1F4-IgG1a-P238S. Compared to the 1F4-CT molecule, 1F4-IgG1a-P238K exhibited weaker FcgR binding, improved thermal stability, and superior heterogeneity. Thus, the single P238K mutation unexpectedly provided all three desired properties when designing this set of hinge / Fc variants: equivalent or weaker FcgR binding, superior thermal stability, and reduced heterogeneity compared to 1F4-CT.

[0172] The 1F4-N297A molecule exhibited lower thermal stability of the CH2 domain and weaker FcgR binding compared to 1F4-IgG1f, characteristics consistent with literature reports for other IgG1 antibodies containing the N297A mutation. The homogeneity of 1F4-N297A was similar to that of 1F4-IgG1f.

[0173] Overall, the 1F4-IgG molecules that exhibited the weakest FcgR binding were the 1F4-IgG1a-P238K, 1F4-N297A, and 1F4-CT molecules. Of these, 1F4-IgG1a-P238K and 1F4-N297A had superior thermal stability and homogeneity compared to 1F4-CT, and 1F4-IgG1a-P238K had superior thermal stability than 1F4-N297A. As a result, the P238K and N297A isotypes were selected as leads for further characterization.

[0174] Example 6: Characterization of dAb-Fc antibodies containing variant Fc domains The SPR, DSC, icIEF and MS data for FcgR binding of the 1F4-IgG molecule provided considerable insight into the regions and mutations of the CT isotype that contribute to FcgR binding, stability and heterogeneity, as discussed in Example 5. These data were therefore used to prioritize a subset of dAb-Fc isotype variants for expression as small-scale expression supernatants for screening by SPR for FcgR binding.

[0175] For example, the P238K and N297A single mutants of the 1F4 antibody exhibited advantageously weaker FcγR binding properties while maintaining superior thermal stability and homogeneity compared to the CT isotype molecule. Therefore, the 3h56-269-IgG1a-C220S,P238K and 3h56-269-IgG1f-C220S,N297A molecules were included in the dAb-Fc analysis.

[0176] Furthermore, the superior homogeneity, similar thermostability, and FcgR-binding properties of the C226A, C229A double mutant compared to the C226S, C229S double mutant raises the possibility that the C226A, C229A double mutant combined with P238S or P238K may have the desired weak FcgR-binding potential without the high heterogeneity and O-linked glycans that result from mutating C226 and C229 to Ser, respectively. As a result, two variants (i.e., the 3h56-269-IgG1a-C220S, C226A, C229A, P238S and 3h56-269-IgG1a-C220S, C226A, C229A, P238K variants) were selected for further investigation.

[0177] The C220 residue in the 1F4-IgG1 molecule was retained as a wild-type Cys to ensure a natural disulfide bond with the antibody light chain, and therefore the effect of mutations at position 220 was not examined for the 1F4-IgG molecule. However, because dAb-Fc antibody polypeptides lack a light chain, the C220 residue either forms a free Cys or forms a potential disulfide bond with another free Cys (such as the C220 residue of a partner dAb-Fc chain). Therefore, a subset of C220 mutants was included in the dAb-Fc variant analysis to determine the effect of mutations at this position on the FcgR-binding properties of the molecule.

[0178] For comparison, a L234A, L235A (LALA) double mutant was generated for both the IgG1a and IgG1f allotypes.

[0179] In addition to the methods described above, the methods used in this example include the following:

[0180] Inhibition of CD40L-induced human B cell proliferation: Human tonsillar B cells were obtained from pediatric patients during routine tonsillectomy, the tissue was minced and gently ground, the cells were passed through a screen, and the cells were incubated with human lympholyte. (登録商標) Mononuclear cells were isolated by density gradient separation using -H separation media (Cedarlane Labs, Burlington, ON). Mononuclear cells were collected from the interface, washed, and rosetted with sheep red blood cells (SRBC, Colorado Serum Company; Denver, CO) for 1 hour at 4°C, followed by density gradient separation to remove T cells. Cells were washed again and resuspended in RPMI containing 10% FBS (complete medium). Antibody titrations were performed in complete medium and added in triplicate to a 96-well round-bottom (RB) plate. 1x10 5Human tonsillar B cells were added and stimulated with either soluble IZ-hCD40L (2 μg / mL) or Chinese hamster ovary cells stably transfected with human CD40L (CHO-hCD40L) irradiated at 10,000 rad, in a final volume of 200 μL per well. 3 Plates were incubated at 37°C and 5% CO2 for 72 hours and 0.5 μCi of IgG per well was added. 3 B cells were labeled with [H]-thymidine for the final 6 hours, harvested, and counted by liquid scintillation. B cell proliferation was quantified based on thymidine incorporation.

[0181] Results - SPR: Selected dAb-Fc variants were expressed as supernatants on a small scale and assayed against immobilized Protein A Biacore as described in Example 2. (商標) The antibodies were captured on an SPR sensor chip surface and tested for binding to purified FcgR analytes (μM). The data are shown in Table 28. [Table 29]

[0182] The SPR data for FcgR binding of the 3h56-269-IgG1a-C220S variant were similar to those of 3h56-269-IgG1a, suggesting that the C220S mutation has only a minor effect on FcgR binding. However, this mutation may be preferable for potential development in a dAb-Fc format, as it removes a potentially reactive thiol group that could pose a risk for heterogeneity during manufacturing or storage.

[0183] The SPR data for FcgR binding of other dAb-Fc molecules were in good agreement with those of the 1F4-IgG variants. For example, all variants containing P238K or N297A showed weaker hCD64 binding compared to the wild-type, and all other FcgR binding was essentially undetectable. The P238K and N297A variants showed weaker hCD64 binding than 3h56-269-CT, similar to that observed for the analogous 1F4-IgG variants. Similar to the 1F4-IgG variants, the single P238S mutation or the C226S / C229S double mutation reduced FcgR binding, but not as much as the combination of these three mutations (3h56-269-CT). Furthermore, a mutant in which both hinge Cys residues were mutated to Ala (3h56-269-IgG1a-C220S,C226A,C229A) exhibited FcgR binding similar to that of the double hinge Cys-to-Ser variant (3h56-269-IgG1a-C220S,C226S,C229S). Addition of the P238S mutation further reduced FcgR binding, similar to that observed with the 1F4-IgG molecule (3h56-269-IgG1a-C220S,C226A,C229A,P238S).

[0184] The LALA variants tested had significantly reduced FcgR binding, especially compared to wild-type, and exhibited the weakest hCD64 binding of any variant tested, but they did exhibit stronger hCD16a-V158 binding than either 3h56-269-CT or the P238K or N297A molecules.

[0185] Based on the SPR data obtained using the dAb-Fc supernatant, the dAb-Fc variants with the weakest binding to low-affinity FcgR were selected for purification and further characterization. These variants included 3h56-269-IgG1a-C220S, C226A, C229A, P238S, 3h56-269-IgG1a-C220S, C226A, C229A, P238K, 3h56-269-IgG1a-C220S, P238K, and 3h56-269-IgG1f-C220S, N297A. All four molecules were shown to bind with high affinity to the CD40 target using SPR. The data are shown in Table 29. [Table 30]

[0186] Binding of purified dAb-Fc to FcgR was assessed by SPR as described in Example 2. Data for dAb-Fc and the 1F4 antibody control are shown in Table 30. [Table 31]

[0187] The SPR data for purified dAb-Fc (along with the 1F4 antibody control) was consistent with the data for the dAb-Fc supernatant, indicating that the CT, N297A, and P238K variants bound weakest to the low-affinity FcgR. See Table 30. This trend was consistent in both the 1F4 antibody and dAb-Fc formats. Indeed, in the dAb-Fc format, at the highest concentrations tested, 3h56-269-IgG1a-C220S, P238K, 3h56-269-IgG1a-C220S, C226A, C229A, P238K, and 3h56-269-IgG1f-C220S, N297A all showed even weaker FcgR binding responses than 3h56-269-CT.

[0188] Results—iDC Activation: The 3h56-269-IgG1a-C220S, P238K, and 3h56-269-IgG1f-N297A molecules were tested for their ability to activate iDCs alone or with CD32-mediated clustering / crosslinking, as described in Example 1. The data indicate that these mutations in the IgG1 Fc tail can eliminate any iDC activation, rendering the anti-CD40 dAb-Fc molecules inactive in these iDC activation assays. See Figure 9. Activation of iDCs, as measured by cytokine production and upregulation of CD86 and CD54, was not observed with either the fusion protein alone or with CD32-mediated clustering, highlighting the potential of these mutations to generate CD40 antagonists lacking immunostimulatory potential. In these same donors, a small increase in at least one measure of iDC activation was observed in iDCs from two of the six samples tested when stimulated with 3h-59-269-IgG4.1 alone, and in iDCs from all six donors when CD32-mediated clustering / crosslinking was included.

[0189] Results—Inhibition of CD40L-induced human B cell proliferation: Despite the differential activities of fusion proteins with various Fc tails, these variations do not affect their ability to inhibit CD40L-mediated activation of immune cells (such as B cells). This is exemplified by the activity of the 3h-59-269-IgG1a-P238K and 3h-59-269-IgG1f-N297A fusions. B cell proliferation stimulated with both soluble CD40L trimers and CD40L-expressing CHO cells is similarly potently inhibited by 3h-59-269-IgG1-P238K or 3h-59-269-IgG1-N297A (Table 31). [Table 32]

[0190] Results - DSC: The thermal stability of the four purified dAb-Fcs that exhibited low FcgR binding was characterized by DSC as described in Example 3. Similar to the previously characterized IgG1-type dAb-Fc molecules, all four new molecules exhibit a transition near 83°C characteristic of the CH3 domain of the human IgG1 Fc domain, with lower temperature transitions assigned to the dAb and CH2 domains. These data are in Table 32. See also Figure 11. [Table 33]

[0191] The lower temperature transitions of both the 3h56-269-IgG1a-C220S,C226A,C229A,P238S and 3h56-269-IgG1a-C220S,C226A,C229A,P238K variants are seen at a low T around 40 °C. onset , and had a broad unfolding transition with a Tm1 value of 55.7–55.8 °C, similar to previous data observed for 3h56-269-CT. The thermal stability of 3h56-269-IgG1a-C220S,P238K and 3h56-269-IgG1f-C220S,N297A was similar to previous data observed for 3h56-269-CT. onset , and were very good with Tm1 values ​​of 60.5°C (3h56-269-IgG1f-C220S, N297A) and 61.5°C (3h56-269-IgG1a-C220S, P238K).

[0192] Results—Accelerated Stability Study: The physical stability of the dAb-Fc molecules was studied under accelerated stress conditions. First, a study was conducted to directly compare the physical stability of four optimized new variants (3h56-269-IgG1a-C220S, C226A, C229A, P238S; 3h56-269-IgG1a-C220S, C226A, C229A, P238K; 3h56-269-IgG1a-C220S, P238K; and 3h56-269-IgG1f-C220S, N297A) with the original 3h56-269-CT molecule. Here, samples were prepared at 15 mg / ml in 20 mM acetic acid, 250 mM sucrose, pH 5.0, and incubated at 40°C for 4 weeks. Aliquots were removed at the start of the study (time zero, t0), week 1, and week 4 and subjected to analytical SEC analysis (aSEC). The data are shown in Table 33. [Table 34]

[0193] These data showed a large increase in HMW species (e.g., from 0.5% to over 13%) for 3h56-269-IgG1a-C220S,C226A,C229A,P238S, 3h56-269-IgG1a-C220S,C226A,C229A,P238K, and 3h56-269-CT, and a small increase (e.g., from 0% to 1.2%) for 3h56-269-IgG1a-C220S,P238K and 3h56-269-IgG1f-C220S,N297A.

[0194] To further compare the physical stability of the four optimized dAb-Fc proteins, a second study was performed at higher concentrations. Samples were prepared in 20 mM acetic acid, 250 mM sucrose, pH 5.0 at 70 mg / ml for 3h56-269-IgG1a-C220S,C226A,C229A,P238K, 3h56-269-IgG1a-C220S,P238K, and 3h56-269-IgG1f-C220S,N297A, and at 30 mg / ml for 3h56-269-IgG1a-C220S,C226A,C229A,P238S (the latter sample was at a lower concentration due to limited material resulting from low expression levels and low yields after purification). They were incubated at either 40°C, 25°C, or refrigerated (4°C) temperatures for 4 to 12 weeks, and aliquots were removed at various time points and subjected to analytical SEC analysis. The data are shown in Table 34. [Table 35]

[0195] These data showed a much greater increase in HMW species for 3h56-269-IgG1a-C220S,C226A,C229A,P238S and 3h56-269-IgG1a-C220S,C226A,C229A,P238K compared to 3h56-269-IgG1a-C220S,P238K and 3h56-269-IgG1f-C220S,N297A. The increase in HMW was similar for 3h56-269-IgG1a-C220S,P238K and 3h56-269-IgG1f-C220S,N297A at each of the three temperatures tested.

[0196] Additional control dAb-Fc molecules were generated that altered and enhanced FcgR binding properties, including one containing the wild-type IgG1f Fc domain (3h56-269-IgG1f), or with additional point mutations that enhanced binding to hCD32a-R131 and hCD32b (3h56-269-IgG1-S267E) or specificity for hCD32b (3h56-269-IgG1f-G237D, P238D, H268D, P271G, A330R, also referred to as 3h56-269-IgG1-V11). See sequences 131-133 in Table 35. [Table 36]

[0197] These dAb-Fc were tested for binding to human FcgR using SPR. The data demonstrated the expected binding specificity. See Table 36. [Table 37]

[0198] iDC activation data for 3h-59-269-IgG1-V11 and 3h-59-269-S267E show robust iDC activation at all concentrations tested, both in the absence and presence of CD32-expressing CHO cells (see Figure 10). The ability to modulate immune cell activation is demonstrated by the activity of the 3h56-269-IgG1f fusion, which shows only modest activation in the absence of CD32-mediated crosslinking and increases with CD32-overexpressing CHO cells (see Figure 10).

[0199] Although the present embodiments have been described in detail with reference to the examples above, it will be understood that various modifications can be made without departing from the spirit of these embodiments and will be readily apparent to those skilled in the art.

Claims

1. An antibody polypeptide comprising a human IgG1 Fc domain polypeptide, wherein the human IgG1 Fc domain polypeptide has (i) a Kabat mutation at position 238 that reduces binding to an Fc gamma receptor, wherein proline at position 238 (P238) is mutated to lysine, wherein the antibody polypeptide binds to CD40, and wherein the antibody polypeptide has reduced binding to the Fcg receptor hCD16b-NA2 (FcgRIIIb-NA2), and (ii) the following additional mutations: (1) C220S, C226, and C229 (“SCC”) (2) C220S, C226A, and C229A (“SAA”) (3) C220S, C226A, and C229 (“SAC”), or (4) C220S, C226, and C229A (“SCA”) An antibody polypeptide comprising one of:

2. The further selective mutation is (1) C220S, C226, and C229 (“SCC”), or (2) C220S, C226A, and C229A (“SAA”) The antibody polypeptide of claim 1, selected from:

3. Further selective mutations (1) C220S, C226A, and C229 (“SAC”), or (2) C220S, C226, and C229A (“SCA”) The antibody polypeptide of claim 1, selected from:

4. The antibody polypeptide described in claim 1, wherein the antibody polypeptide further has reduced binding to at least one Fcg receptor selected from hCD32a-H131 (FcgRIIa-H131) and hCD32a-R131 (FcgRIIa-R131).

5. A single variable domain, said single variable domain comprising: (a) a CDR1 region consisting of the amino acid sequence of SEQ ID NO: 1; (b) a CDR2 region consisting of the amino acid sequence of SEQ ID NO: 2; and (c) a CDR3 region consisting of the amino acid sequence of SEQ ID NO: 3; and The antibody polypeptide of claim 1, wherein the single variable domain binds to CD40.

6. An antibody polypeptide as described in claim 5, wherein the single variable domain antagonizes at least one CD40 activity.

7. The antibody polypeptide of claim 5, having increased stability compared to a reference polypeptide comprising the same single variable domain as defined in claim 5 and a wild-type IgG Fc domain.

8. An antibody polypeptide described in claim 5, wherein the amino acid sequence of the single variable domain is set forth in SEQ ID NO: 41 (3h-56-269 sequence).

9. A nucleic acid encoding an antibody polypeptide described in any one of claims 1 to 8.

10. An expression vector comprising the nucleic acid described in claim 9.

11. An isolated cell transformed with the expression vector described in claim 10.

12. A pharmaceutical composition for treating or preventing an immune disease in a subject, comprising: a) an antibody polypeptide described in any one of claims 1 to 8; and b) a pharmaceutically acceptable carrier, wherein the immune disease is selected from the group consisting of Crohn's disease, diabetes, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, rheumatic fever, rheumatoid arthritis, and Sjogren's syndrome.

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