Hinge-modified antibody fragments and methods of making

Abstract

To provide new Fab and F (ab') 2 molecules having reduced or no reactivity with existing anti-hinge antibodies (AHA) in human serum.SOLUTION: In certain embodiments, the present disclosure is directed to isolated antibody fragments and compositions comprising the same, wherein the antibody fragments have reduced or no reactivity to pre-existing anti-hinge antibodies. In certain embodiments, an isolated antibody fragment of the present disclosure exhibits reduced and / or no binding to FcyRIIIa and / or CIq. In certain embodiments, the antibody fragment is an Fab, Fab' or F (ab') 2.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 248,792, filed October 30, 2015, and U.S. Provisional Patent Application No. 62 / 346,905, filed June 7, 2016, the contents of each of which are incorporated herein by reference in their entirety to each of which priority is claimed.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format, and is incorporated herein by reference in its entirety. The ASCII copy, created on October 26, 2016, is named 00B206_0271_SL.txt and is 26,185 bytes in size.

[0003] The present disclosure relates to antibody fragments (e.g., Fab and F(ab')2) that have reduced or no reactivity with existing anti-hinge antibodies (AHAs), compositions comprising such antibody fragments, and methods of making and using such antibody fragments and compositions. [Background technology]

[0004] Antibodies are composed of two Fab regions connected to an Fc by a flexible hinge region. While the Fab mediates antigen recognition and binding, two important functions of the Fc are mediating effector functions by binding to Fcγ receptors (1) and conferring a long serum half-life by binding to the salvage receptor FcRn (2). In particular, the slow pharmacokinetics of IgG contributes to the success of antibodies as therapeutics, as it allows for less frequent dosing compared to other biotherapeutics. As a result, most approved therapeutic antibodies are in the full-length IgG format. Unlike IgG, isolated Fab fragments have a short serum half-life (3), a property required for indications where a short plasma half-life is desirable, as is the case for the three Fab molecules approved by the FDA (4). One therapeutic Fab molecule (abciximab, REOPRO®) directed against the platelet surface receptor GPIIb / IIIa is commercially produced by proteolytic cleavage with papain (5), the original method for Fab production (6). With advances in molecular cloning, recombinant expression of antibody fragments has become an attractive route for generating Fab molecules, as exemplified by the second approved Fab therapeutic, an anti-VEGF drug (ranibizumab, Lucentis®) (7) and the recently approved Fab against dabigatran (idarucizumab, Praxbind®) (33). Fab molecules are advantageous, for example, when transient systemic activity that does not persist after dosing is desired, or when administration and activity are limited to peripheral compartments, such as the eye.

[0005] Many proteases directed against antibody hinge regions have been implicated as a mechanism by which pathogens and tumor cells attempt to evade the host immune response (13). The resulting C-terminal neoepitopes, however, are eventually recognized by the immune system, resulting in the generation of anti-hinge antibodies (AHA). Several studies have demonstrated the presence of such autoantibodies directed against the upper hinge region of Fab and the lower hinge region of F(ab')2 (17-21). These pre-existing AHA titers vary between donors (20), which may represent past and current exposure to such neoepitopes. In certain instances, AHA can act as a surrogate Fc to restore effector function to proteolytically inactivated antibodies (22). Because one rationale for using Fab or F(ab')2 molecules as therapeutic formats is to eliminate effector function, it would be undesirable to restore effector function with pre-existing AHA and risk any potential safety concerns. Therefore, there is a need in the art for, inter alia, novel Fab and F(ab')2 molecules with reduced or no reactivity with existing AHAs in human serum, which may offer a better safety profile in the therapeutic setting by minimizing immune responses following drug treatment. Summary of the Invention

[0006] The present disclosure relates to antibody fragments (e.g., Fab and F(ab')2) that have reduced or no reactivity with existing anti-hinge antibodies (AHAs), compositions comprising such antibody fragments, and methods of making and using such antibody fragments and compositions.

[0007] In certain embodiments, the present disclosure is directed to isolated antibody fragments and compositions comprising the same, which have reduced or no reactivity with pre-existing anti-hinge antibodies. In certain embodiments, the isolated antibody fragments of the present disclosure exhibit reduced and / or no binding to FcγRIIIa and / or C1q. In certain embodiments, the antibody fragment is a Fab, Fab', or F(ab')2.

[0008] In certain embodiments, the present disclosure is directed to antibody fragments and compositions comprising the same, wherein the antibody fragment is a Fab. In certain embodiments, the present disclosure is directed to a Fab molecule, wherein the Fab is a Fab having residues D 221 In certain embodiments, the Fab terminates with an amino acid comprising an amino acid sequence selected from the group consisting of CDKTHT (SEQ ID NO: 14), CDKTHL (SEQ ID NO: 15), CDKTH (SEQ ID NO: 16), CDKT (SEQ ID NO: 17), CDK, and CD. In certain embodiments, the Fab terminates with an amino acid comprising an amino acid sequence selected from the group consisting of KYGPP (SEQ ID NO: 18), KYGP (SEQ ID NO: 19), KYG, KY, and K. In certain embodiments, the Fab comprises a heavy chain constant region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and conservative modifications thereof.

[0009] In certain embodiments, the present disclosure is directed to antibody fragments and compositions comprising the same, wherein the antibody fragment is F(ab')2. In certain embodiments, the present disclosure is directed to F(ab')2 molecules, wherein the F(ab')2 comprises a deletion of 1, 2, 3, 4, or 5 amino acids at the C-terminus. In certain embodiments of the present disclosure, the F(ab')2 comprises a deletion at EU position 231. In certain embodiments of the present disclosure, the F(ab')2 comprises a deletion at EU positions 231-232. In certain embodiments of the present disclosure, the F(ab')2 comprises a deletion at EU positions 231-233. In certain embodiments of the present disclosure, the F(ab')2 comprises a deletion at EU positions 231-234. In certain embodiments, the F(ab')2 comprises a deletion at EU positions 230-234.

[0010] In certain embodiments, the present disclosure is directed to an isolated nucleic acid and a composition comprising the same, wherein the nucleic acid encodes an antibody fragment with reduced or no reactivity to AHA. In certain embodiments, the present disclosure is directed to a host cell comprising the nucleic acid. In certain embodiments, the present disclosure is directed to a method for producing an antibody fragment, comprising culturing the host cell to produce an antibody. In certain embodiments, the present disclosure is directed to a pharmaceutical formulation comprising an antibody fragment with reduced or no reactivity to AHA and a pharmaceutically acceptable carrier.

[0011] In certain embodiments, the present disclosure is directed to antibody fragments with reduced or no reactivity to AHA for use as medicaments. In certain embodiments, the present disclosure is directed to antibody fragments with reduced or no reactivity to AHA for use in treating a disease. In certain embodiments, the present disclosure is directed to antibody fragments with reduced or no reactivity to AHA for use in inhibiting or activating a molecular pathway and / or mechanism. In certain embodiments, the present disclosure is directed to the use of antibody fragments with reduced or no reactivity to AHA in the manufacture of a medicament for treating a disease. In certain embodiments, the present disclosure is directed to the use of antibody fragments with reduced or no reactivity to AHA in the manufacture of a medicament for inhibiting or activating a molecular pathway and / or mechanism.

[0012] In certain embodiments, the present disclosure is directed to methods of treating an individual having a disease, comprising administering to the individual an effective amount of an antibody fragment with reduced or no reactivity to AHA. In certain embodiments, the present disclosure is directed to methods of inhibiting or activating a molecular pathway and / or mechanism in an individual, comprising administering to the individual an effective amount of an antibody fragment with reduced or no reactivity to AHA to inhibit or activate the molecular pathway and / or mechanism. [Brief explanation of the drawings]

[0013] [Figure 1]Figures 1A-1D show the binding of pre-existing human antibodies to human IgG1, IgG2, and IgG4 Fabs. (1A) X-ray crystal structure of the Fab region (PDB: 1HZH) including the upper hinge: light chain (101), heavy chain (102), interchain disulfide (103), and upper hinge (104). In isolated Fab molecules, the upper hinge is a protruding, unstructured region with no structural or functional role. Residues in the upper hinge are colored magenta to indicate the T225L mutation (105), which disrupts pre-existing binding to AHA. Residue numbering follows the EU nomenclature. Figure 1A discloses SEQ ID NOS: 14-15 in order of appearance, respectively. (1B) Pooled human sera were incubated with human IgG1 Fabs with different upper hinge lengths and termini. Pre-existing bound antibodies were detected by anti-Fc ELISA. Truncation at D221 (D) of the Fab C-terminus and the C-terminal mutant T225L (DKTHL (SEQ ID NO: 20)) significantly reduced binding of the pre-existing antibody to nearly background. A strong response was observed with T223 (DKT) as the C-terminal residue, which coincides with the cleavage site of human neutrophil elastase. The average values ​​of the individual data points are represented by horizontal lines. Figure 1B discloses SEQ ID NOs: 20-21 and 27, respectively, in order of appearance. (1C) Three different Fabs were incubated with pooled human serum, and pre-existing antibody binding was detected by ELISA. Significant signals were observed for the different Fabs with the DKTHT (SEQ ID NO: 21) C-terminus. Reduced binding of the pre-existing antibody to the D221 and T225L C-termini was detected across the different Fabs. Fab-1 contains the antibody variable domain used in (B) and all other AHA binding experiments throughout Example 1. Figure 1C discloses SEQ ID NOs: 21, 20, 21, 20, 21, and 20, respectively, in order of appearance. (1D) Pooled human serum was incubated with human IgG2 Fab and IgG4 Fab with different upper hinge lengths, and bound antibodies were detected by ELISA. No pre-existing antibodies to the upper hinges of human IgG2 and IgG4 were detectable. Figure 1D discloses SEQ ID NO: 18. [Figure 2]Figures 2A-2C show the cleavage of an IgG1-2 chimera by IdeS. (2A) Model of the F(ab')2 region of antibody cAC10 modeled with MOE: light chain (201), heavy chain (202), interchain disulfide (203), and lower hinge (204). The P1 position of IdeS is G236. Residue numbering follows the EU nomenclature. Figure 2A discloses SEQ ID NO: 30. (2B) Alignment of the lower hinges of IgG1 and IgG1-2 chimeras. Cyan residues are IgG2 isotype residues introduced into the lower hinge of IgG2. Figure 2B discloses SEQ ID NOs: 31 and 59, respectively, in order of appearance. (2C) Cleavage efficiency of human IgG1 and IgG1-2 chimeras. 1 mg / ml IgG1 and IgG1-2 were incubated with the indicated amounts of IdeS at 37°C for 24 hours. Cleavage was analyzed by capillary electrophoresis. IgG1 was efficiently cleaved to F(ab')2 at an IdeS:IgG ratio of 1:500, while IgG1-2 required a 50-fold higher IdeS concentration for complete cleavage. [Figure 3A-B] Figures 3A-3E show the cleavage of human IgG1 with mutations at the P1 and P2 positions by IdeS. (3A) Capillary electrophoresis of antibodies with mutations at the P1 and P2 positions was performed at 37°C for 24 hours with a 1:10 ratio of IdeS:IgG at 1 mg / ml. The P1 and P2 residues are indicated by single-letter code. Leucine and glycine (L235G236) are natural amino acids at these positions. All antibody mutants were able to be completely cleaved into F(ab')2 fragments. (3B) The cleavage efficiency of the mutants was assessed by the amount of F(ab')2 produced at different IdeS:IgG ratios. While the VG mutant was cleaved equivalently to the wild-type sequence (LG), the other mutants required increased amounts of IdeS for complete digestion. [Figure 3C] (3C) The cleavage efficiency of the mutants was assessed by the amount of F(ab')2 produced at an IdeS:IgG ratio of 1:10. [Figure 3D] (3D) Schematic representation of the expression, purification and screening strategy for human IgG1 variants. Figure 3D discloses SEQ ID NOs: 31-34, respectively, in order of appearance. [Figure 3E] (3E) Cleavage efficiency of 76 human IgG1 variants by IdeS. Figure 3E discloses SEQ ID NO: 31. [Figure 4] Figures 4A-4B show the reactivity of the P1 and P2 mutants with pre-existing AHA. (4A) IdeS was efficiently removed during purification and was undetectable in the purified F(ab')2 mutants by SDS-PAGE followed by Coomassie staining (upper panel) or immunoblot analysis with anti-IdeS antibody (lower panel). (4B) Pooled human serum was incubated with a human IgG1 Fab with a T225 C-terminus and an F(ab')2 generated by IdeS cleavage of an antibody with mutations at the P1 and P2 positions. Pre-existing bound antibodies were detected by ELISA. The F(ab')2 showed approximately 1.7-fold higher signal than the Fab. Hinge mutations reduced reactivity to a level comparable to that of the Fab but did not completely eliminate reactivity. [Figure 5A-C] Figures 5A-5F show the reactivity of truncated mutants to pre-existing AHA responses. (5A) IdeS cleavage of antibodies with deletions at IdeS P3, P4, and P5 sites. Deletion of the IdeS P3 residue (L234) in the lower hinge significantly affected cleavage efficiency, while deletion of P4 (E233) or P5 (P232) sites did not affect cleavage by IdeS compared to the wild type (WT). (5B) Deletion of P4 and P5 sites was not sufficient to prevent pre-existing AHA binding. (5C) IdeS cleavage of antibodies with deletions at IdeS P4-P6 (ΔP456) and P4-P7 (ΔP4567) sites. At an IdeS:IgG ratio of 1:200, the cleavage efficiency of the ΔP4567 mutant was slightly reduced compared to the wild-type lower hinge sequence (WT), while ΔP456 showed cleavage efficiency equivalent to that of the wild-type. [Figure 5D-E](5D) Pooled human serum was incubated with F(ab')2 produced by IdeS digestion, and bound antibodies were detected by ELISA. The lower hinge deletions ΔP456 and ΔP4567 were not recognized by existing AHAs. (5E) IdeS cleaved the ΔP456 hinge mutant with high specificity. After digestion of wild-type (WT) and ΔP456 hinge IgG, the reduced F(ab')2 was analyzed by mass spectrometry. Only a single heavy chain species corresponding to the expected molecular weight was observed. [Figure 5F] (5F) Schematic diagram illustrating the deletions made in the lower hinge region. Figure 5F discloses SEQ ID NOS: 35, 31, 36-40, 31, and 41-42, respectively, in order of appearance. [Figure 6] Figures 6A-6B show an alignment of amino acid residues (6A) and EU numbering of amino acid residues (6B) in the upper, core, and lower hinge regions of human, cynomolgus, and rhesus IgG1, IgG2, IgG3, and IgG4 isotypes. Figure 6A discloses SEQ ID NOS: 43-54, respectively, in order of appearance. Figure 6B discloses SEQ ID NOS: 43, 46, 55, and 52, respectively, in order of appearance. [Figure 7] 1 shows the expression levels of Fabs with upper hinge truncations or mutations in E. coli. The figure discloses SEQ ID NOS: 14 to 15 in order of appearance, respectively. [Figure 8] 1 shows the efficiency of cleavage of ΔP456 and ΔP4567 mutants produced at an IdeS:IgG ratio of 1:500 or 1:10. The figure discloses SEQ ID NOs:56 to 58, respectively, in order of appearance. [Figure 9A] Figures 9A-9D show the reactivity of deletion mutants with modified P1 and P2 residues with pre-existing AHA. (9A) The cleavage efficiency of the mutants was assessed by the amount of F(ab')2 produced at an IdeS:IgG ratio of 1:10. Figure 9A discloses SEQ ID NO: 31. [Figure 9B] (9B) The cleavage efficiency of the mutants was assessed by the amount of F(ab')2 produced at an IdeS:IgG ratio of 1:100. Figure 9B discloses SEQ ID NO:31. [Figure 9C](9C) The cleavage efficiency of the mutants was assessed by the amount of F(ab')2 produced at an IdeS:IgG ratio of 1:500. Figure 9C discloses SEQ ID NO:31. [Figure 9D] (9D) Detection of pre-existing AHA bound to the mutant by anti-Fc ELISA. Figure 9D discloses SEQ ID NO: 56. [Figure 10A] Figures 10A-10B show the reactivity of ΔP456 and ΔP4567 mutants with modified P1 and P2 residues to pre-existing AHA. (10A) The cleavage efficiency of the mutants was assessed by the amount of F(ab')2 produced at IdeS:IgG ratios of 1:10 and 1:200. Figure 10A discloses SEQ ID NOS:56-57, respectively, in order of appearance. [Figure 10B] (10B) Detection of pre-existing AHA bound to the mutant by anti-Fc ELISA. Figure 10B discloses SEQ ID NO: 56. [Figure 11] Figure 1 shows the titration curves of F(ab')2 and Fab molecules in AHA ELISA. The dilutions corresponding to OD 450 nm (1.15) at the center of the F(ab')2 titration curve were 70 and 14 for F(ab')2 and Fab, respectively. Therefore, F(ab')2 has 5-fold higher AHA reactivity than IgG1 Fab. F(ab')2, F(ab')2ΔP456, Fab T225, Fab T225L, and Fab D221 were coated onto wells. Serial dilutions of pooled human serum were added to the wells, and control wells were uncoated. Similar results were obtained in four other experiments. The data shown in this figure and in Figures 1B and 5D were collected from the same experiment. The figures disclose SEQ ID NOs: 21 and 20, respectively, in order of appearance. DETAILED DESCRIPTION OF THE INVENTION

[0014] I. Definition The term "antibody" as used herein is used in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0015] "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments. In certain embodiments, the antibody fragment is a Fab molecule. In certain embodiments, the antibody fragment is a F(ab')2 molecule.

[0016] The terms "full-length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody having a heavy chain that has a structure substantially similar to a native antibody structure or that contains an Fc region as defined herein.

[0017] "Native antibodies" refer to naturally occurring immunoglobulin molecules with a variety of structures. For example, native IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 daltons, composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From the N-terminus to the C-terminus, each heavy chain contains a variable region (VH), also called a variable heavy domain or heavy chain variable domain, followed by three constant domains (C H 1. C H 2, and C H 3). Similarly, from N- to C-terminus, each light chain has a variable region (VL), also called a variable light domain or light chain variable domain, followed by a constant light (CL) domain. The light chain of an antibody can be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain.

[0018] The term "Fc region" is used herein to define a C-terminal region of an immunoglobulin heavy chain containing at least a portion of a constant region. This term includes native sequence Fc regions and variant Fc regions. Unless otherwise specified herein, numbering of amino acid residues within an Fc region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0019] "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. FcγRII receptors include FcγRIIA (an "activating receptor") and FcγRIIB (an "inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain (see, e.g., Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed, for example, in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991), Capel et al., Immunomethods 4:25-34 (1994), and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including those identified in the future, are encompassed by the term "FcR" herein.

[0020] The term "Fc receptor" or "FcR" also includes the neonatal receptor FcRn, which is involved in the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) and in regulating immunoglobulin homeostasis. Methods for measuring binding to FcRn are known (see, e.g., Ghetie and Ward, Immunol. Today 18(12):592-598 (1997); Ghetie et al., Nature Biotechnology, 15(7):637-640 (1997); Hinton et al., J. Biol. Chem. 279(8):6213-6216 (2004); WO2004 / 92219 (Hinton et al.)).

[0021] In vivo binding to human FcRn and serum half-life of human FcRn high-affinity binding polypeptides can be assayed, for example, in transgenic mice or transfected human cell lines expressing human FcRn, or in primates administered with polypeptides having mutant Fc regions. WO 2000 / 042072 (Presta) describes antibody variants with improved or reduced FcR binding. See, e.g., Shields et al. J. Biol. Chem. 9(2):6591-6604 (2001).

[0022] "Effector function" refers to a biological activity attributable to the Fc region of an antibody, which varies depending on the antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, down-regulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0023] The "hinge region" is generally defined as the section from 216 to 238 (EU numbering) or 226 to 251 (Kabat numbering) of human IgG1. The hinge can be further divided into three distinct regions: the upper, middle (e.g., core), and lower hinge. See, e.g., Brezski and Georgiou, Curr. Opin. Immunol. 40, 62-69 (2016), which is incorporated herein by reference in its entirety. In certain embodiments, the hinge region of a human IgG1 antibody is generally defined as follows:

[0024] The upper hinge comprises amino acids having the sequence EPKSCDKTHT (SEQ ID NO: 22). In certain embodiments, the upper hinge comprises amino acids 216-225 (EU numbering) or 226-238 (Kabat numbering).

[0025] The middle (e.g., core) hinge comprises amino acids having the sequence CPPC (SEQ ID NO: 23). In certain embodiments, the core hinge comprises amino acids 226-229 (EU numbering) or 239-242 (Kabat numbering).

[0026] The lower hinge comprises amino acids having the sequence PAPELLGGP (SEQ ID NO: 24). In certain embodiments, the lower hinge comprises amino acids 230-238 (EU numbering) or 243-251 (Kabat numbering).

[0027] The "class" of an antibody refers to the type of constant domain or constant region carried by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0028] An "isolated" antibody or antibody fragment is an antibody that has been separated from the components of its natural environment.Antibodies or antibody fragments can be purified to greater than 95% or 99% purity, as determined, for example, by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse-phase HPLC).For a review of methods for assessing antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0029] "Percent amino acid sequence identity (%)" to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence, after aligning the sequences and introducing gaps as necessary to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for determining percent amino acid sequence identity can be achieved in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for sequence alignment, including any algorithms required to achieve maximum alignment across the entire length of the sequences being compared. However, for purposes herein, percent amino acid sequence identity values ​​are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was created by Genentech, Inc., and its source code, together with user documentation, has been filed with the U.S. Copyright Office (Washington DC, 20559), where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or can be compiled from the source code. The ALIGN-2 program should be compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

[0030] In situations where ALIGN-2 is used for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to, with, or relative to a given amino acid sequence B (which may alternatively be expressed as a given amino acid sequence A having or containing a certain % amino acid sequence identity to, with, or relative to a given amino acid sequence B) is calculated as follows: 100 x fraction X / Y where X is the number of amino acid residues scored as perfect matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and Y is the total number of amino acid residues in B. It is understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, then the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless specifically indicated otherwise, all % amino acid sequence identity values ​​used herein are obtained using the ALIGN-2 computer program as described in the immediately preceding paragraph.

[0031] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. Isolated nucleic acid includes a nucleic acid molecule that is contained in a cell that ordinarily contains the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0032] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors as self-replicating nucleic acid structures as well as vectors that have integrated into the genome of a host cell into which they have been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0033] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and its progeny regardless of the number of passages. The progeny may be completely identical in nucleic acid content to the parent cell, or may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0034] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

[0035] As used herein, "treatment" (and grammatical variations thereof, such as "treat" or "treating") refers to a clinical intervention aimed at altering the natural course of the individual being treated and may be performed prophylactically or during the clinical pathology process. Desirable effects of treatment include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction in the rate of disease progression, amelioration or remission of disease symptoms, and remission or improved prognosis. In certain embodiments, the antibody fragments of the present disclosure are used to delay the onset of disease or slow the progression of disease.

[0036] An "effective amount" of an agent, e.g., an antibody fragment described herein, or a pharmaceutical formulation containing the agent, refers to an amount effective, at the necessary dosages and for the necessary periods of time, to achieve the desired therapeutic or prophylactic result.

[0037] The term "package insert" is used to refer to instructions typically included in commercial packaging for therapeutic products that contain information about the indications, uses, dosages, administration, concomitant therapies, contraindications, and / or warnings regarding the use of such therapeutic agent.

[0038] The term "pharmaceutical formulation" refers to a preparation in which the biological activity of the active ingredient contained therein is in a form such that it is effective, and which does not contain any additional components that are unacceptably toxic to the subject to which the formulation is administered.

[0039] A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0040] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents cell function and / or causes cell death or destruction. Cytotoxic agents include radioisotopes (e.g., At 211 , I 131 , I 125 , Y 90 ,Re 186 ,Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 and radioactive isotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin, or other intercalating agents); growth inhibitory agents; enzymes and fragments thereof, such as nucleolytic enzymes; antibiotics; toxins, such as small molecule toxins or enzymatically active toxins (including fragments and / or variants thereof) of bacterial, fungal, plant, or animal origin; and various anti-tumor or anti-cancer agents described below.

[0041] As used herein, the term "about" or "approximately" means within an acceptable error range for a particular value, as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 3 or more than 3 standard deviations, per practice in the art. Alternatively, "about" can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and even more preferably up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold of a value.

[0042] II. Compositions and Methods In certain embodiments, the present disclosure is based, in part, on methods for engineering antibody fragments to avoid pre-existing anti-hinge antibodies (AHAs). In certain embodiments, antibody fragments (e.g., Fab and F(ab')2) with reduced or no reactivity to AHAs and methods for producing these antibody fragments are provided. In certain embodiments, the antibody fragments of the present disclosure may provide greater safety in therapeutic settings by minimizing immune responses after drug treatment.

[0043] A. Exemplary Antibody Fragments In certain embodiments, the present disclosure provides antibody fragments (e.g., Fab, Fab', and F(ab')2) and compositions comprising same that have reduced or no reactivity to AHA. For example, without limitation, the antibody fragments described herein exhibit AHA reactivity that is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% compared to a reference antibody fragment, e.g., an antibody fragment with a native hinge region. In certain embodiments, the reference antibody fragment is an IgG1 antibody fragment with a native hinge region.

[0044] In certain embodiments, isolated antibody fragments of the present disclosure, and compositions comprising the same, exhibit reduced and / or no binding to FcγRIIIa and / or C1q. For example, and without limitation, antibody fragments of the present disclosure exhibit binding to FcγRIIIa and / or C1q that is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% compared to a reference antibody fragment, e.g., an antibody fragment having a native hinge region. In certain embodiments, the reference antibody fragment is an IgG1 antibody fragment having a native hinge region.

[0045] In certain embodiments, antibody fragments used in the context of the methods described herein comprise a native or modified hinge region. For example, without limitation, an antibody fragment of the present disclosure may be a Fab fragment comprising a native or modified hinge region. In certain embodiments, an antibody fragment of the present disclosure is a F(ab')2 comprising a native or modified hinge region.

[0046] The natural hinge region is usually located at the C H 1 domain is associated with the hinge region. In certain embodiments, the native hinge region of the antibody fragments disclosed herein may be of the IgG1, IgG2, IgG3, or IgG4 isotype. For example, without limitation, the Fab fragment may be of the IgG1, IgG2, IgG3, or IgG4 isotype. In certain embodiments, the antibody fragment, e.g., the Fab fragment, is of the IgG2 isotype, which contains the native hinge region. In certain embodiments, the antibody fragment, e.g., the Fab fragment, is of the IgG4 isotype, which contains the native hinge region.

[0047] A modified hinge region is any hinge that differs in length and / or composition from the native hinge region. Such hinges may include hinge regions from other species, such as human, mouse, rat, rabbit, pig, hamster, camel, llama, or goat hinge regions. Other modified hinge regions include C H The C domain may contain a complete hinge region derived from an antibody of a different class or subclass than that of the C domain. Thus, for example, a C domain of class γ1 H 1 domain may be linked to a hinge region of class γ4. Alternatively, the modified hinge region may comprise a portion of a natural hinge or a repeating unit in which each unit in the repeat is derived from a natural hinge region.

[0048] In certain embodiments, the native hinge region is altered by substituting, deleting, and / or adding one or more amino acid residues to generate a modified hinge region. In certain embodiments, the Fab fragment is of the IgG1 isotype containing a modified hinge region. In certain embodiments, the Fab fragment is of the IgG2 isotype containing a modified hinge region. In certain embodiments, the Fab fragment is of the IgG4 isotype containing a modified hinge region.

[0049] In certain embodiments, the modified hinge region comprises a substitution, deletion, and / or addition of one or more amino acids in the upper hinge region. For example, without limitation, a modified hinge region of the disclosed subject matter can have one or more substitutions, deletions, and / or additions at amino acid positions EU216-EU225. Alternatively or additionally, the modified hinge region comprises a substitution, deletion, and / or addition of one or more amino acids in the lower hinge region. In certain embodiments, a modified hinge region of the disclosed subject matter can have one or more substitutions, deletions, and / or additions at amino acid positions EU230-EU238. Alternatively or additionally, the modified hinge region can comprise the addition of one or more amino acids C-terminal to amino acid position EU238. In certain embodiments, the modified hinge region comprises a substitution, deletion, and / or addition of one or more amino acids in the middle, e.g., core, hinge region. For example, without limitation, a modified hinge region of the disclosed subject matter can have one or more substitutions, deletions and / or additions at amino acid positions EU226-229.

[0050] In certain embodiments, the modification or alteration is a substitution of one or more, two or more, three or more, four or more, five or more, or six or more amino acid residues. In certain embodiments, substitutions may be made in the upper hinge region, middle hinge region, and / or lower hinge region. In certain embodiments, the amino acid residue at position 225 may be substituted. For example, and without limitation, the amino acid residue at position 225 may be changed to any amino acid except threonine (T). In certain embodiments, the amino acid at position 225, e.g., threonine, may be changed to leucine (L) (e.g., T225L according to EU numbering). In certain embodiments, the antibody fragment of the present disclosure is a Fab fragment comprising the substitution T225L.

[0051] In certain embodiments, the upper hinge region of an IgG1 antibody fragment may be substituted with one or more amino acid residues present in the upper hinge region of an IgG2 and / or IgG4 antibody, for example, because the upper hinge regions of IgG2 and IgG4 antibodies exhibit reduced or no reactivity to AHAs (see, e.g., Figure 1). For example, and without limitation, the upper hinge region of an IgG1 antibody fragment may be substituted with one or more amino acid residues present in the native hinge region of an IgG2 and / or IgG4 antibody (see, e.g., Figure 6). In certain embodiments, the modified hinge region of an IgG1 antibody fragment retains a cysteine ​​at amino acid position EU220 (e.g., compared to the native hinge region of an IgG1 antibody). In certain embodiments, the modified hinge region of an IgG1 antibody fragment does not retain the cysteine ​​at amino acid position EU220, for example, in an IgG antibody fragment in which the upper hinge region of an IgG1 antibody fragment has been replaced with the upper hinge region of an IgG4 (e.g., the entire upper hinge region). In certain embodiments, the upper hinge region of an IgG1 antibody fragment may be substituted with one or more amino acid residues present in the upper hinge region of an IgG2, IgG3 and / or IgG4 antibody, wherein the amino acid residue at position 131 of the IgG1 antibody is changed from serine (S) to cysteine ​​(C) (i.e., S131C).

[0052] In certain embodiments, an antibody fragment of the present disclosure, e.g., Fab, F(ab')2, or Fab', may comprise a substitution at amino acid EU positions 235-236. For example, without limitation, the amino acid at position 236, e.g., glycine (G), may be changed to alanine (A) (e.g., G236A). In certain embodiments, an antibody fragment, e.g., F(ab')2, may comprise a substitution at position 235 according to EU numbering. In certain embodiments, the amino acid at position 235, e.g., leucine (L), may be changed to valine (V) (e.g., L235V), isoleucine (I) (e.g., L235I), or methionine (M) (e.g., L235M).

[0053] In certain embodiments, the modification or alteration is a deletion of one or more, two or more, three or more, four or more, five or more, or six or more amino acid residues. In certain embodiments, one or more deletions may be made in the upper hinge region, the middle hinge region, and / or the lower hinge region. In certain embodiments, an antibody fragment of the present disclosure, e.g., Fab, F(ab')2, or Fab', comprises a modified hinge region having one or more deletions of one or more amino acids at positions EU230-EU238. In certain embodiments, an antibody fragment comprises a deletion at EU231. In certain embodiments, an antibody fragment comprises deletions at EU231 and EU232. In certain embodiments, an antibody fragment comprises deletions at EU231, EU232, and EU233. In certain embodiments, an antibody fragment comprises deletions at EU231, EU232, EU233, and EU234. In certain embodiments, the antibody fragment comprises a deletion in EU230, EU231, EU232, EU233 and EU234.

[0054] In certain embodiments, antibody fragments of the present disclosure comprise deletion of one or more, two or more, three or more, four or more, five or more, or six or more amino acids at the C-terminus. In certain embodiments, antibody fragments of the present disclosure comprise deletion of one or more amino acids in the upper hinge region, for example, to generate a C-terminal truncation. In certain embodiments, one or more amino acids in EU222-EU225 can be deleted to obtain a C-terminal truncation. In certain embodiments, antibody fragments described herein, e.g., Fab fragments, comprise a C-terminal truncation. For example, and without limitation, the C-terminus of an antibody fragment described herein, e.g., a Fab fragment, ends at amino acid residue D221 (according to EU numbering). In certain embodiments, the C-terminus of an antibody fragment described herein, e.g., a Fab fragment, ends at amino acid residue K222 (according to EU numbering).

[0055] In certain embodiments, the C-terminus of the heavy chain of an antibody fragment, e.g., a Fab fragment, described herein, terminates with an amino acid having a sequence selected from CDKTHT (SEQ ID NO: 14), CDKTHL (SEQ ID NO: 15), CDKTH (SEQ ID NO: 16), CDKT (SEQ ID NO: 17), CDK, and CD. In certain embodiments, the C-terminus of the heavy chain of a Fab fragment terminates in the amino acid sequence CDKTHX (SEQ ID NO: 25), where X is any amino acid except T. In certain embodiments, a Fab fragment comprises a heavy chain constant region selected from "CDKTHT," (SEQ ID NO: 14), "CDKTHL," (SEQ ID NO: 15), "CDKTH," (SEQ ID NO: 16), "CDKT," (SEQ ID NO: 17), "CDK," or "CD," as disclosed in Table 1. In certain embodiments, the presently disclosed subject matter provides antibody fragments, e.g., Fab fragments, comprising a heavy chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, or 6. In certain embodiments, antibody fragments of the present disclosure comprise a heavy chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 5. In certain embodiments, antibody fragments of the present disclosure comprise a heavy chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 6.

[0056] In certain embodiments, as an alternative to truncation and / or mutation at the C-terminus, IgG2 or IgG4 Fab fragments may be used to circumvent a pre-existing AHA response. For example, without limitation, antibody fragments of the present disclosure may comprise a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 7 or 8. In certain embodiments, IgG2 or IgG4 Fab fragments may comprise deletion of one or more, two or more, three or more, four or more, or five or more amino acids at the C-terminus. In certain embodiments, a Fab of the present disclosure is an IgG2 Fab fragment comprising a heavy chain constant region ending with the sequence VERK (SEQ ID NO: 26). In certain embodiments, the C-terminus of the heavy chain of an antibody fragment described herein, e.g., an IgG4 Fab fragment, terminates with an amino acid having a sequence selected from KYGPP (SEQ ID NO: 18), KYGP (SEQ ID NO: 19), KYG, KY, and K. In certain embodiments, a Fab of the present disclosure is an IgG4 Fab fragment comprising a heavy chain constant region selected from "KYGPP," (SEQ ID NO: 18), "KYGP," (SEQ ID NO: 19), "KYG," "KY," and "K," as disclosed in Table 1. For example, and without limitation, an antibody fragment of the present disclosure can comprise a heavy chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 9, 10, 11, 12, or 13. Table 1 - Fab heavy chain sequences TIFF2026035577000001.tif240170TIFF2026035577000002.tif198170

[0057] The present disclosure further provides antibody fragments comprising the conservatively modified sequences described herein.For example, without limitation, the present disclosure provides antibody fragments comprising heavy chain constant regions comprising the amino acid sequences described in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or conservatively modified versions thereof, and these antibody fragments retain the desired properties of the antibody fragments described herein.For example, without limitation, these antibody fragments have reduced or no reactivity to AHA as disclosed above.

[0058] As used herein, the term "conservative sequence modifications" is intended to refer to amino acid modifications that do not significantly affect the properties of the antibody fragment containing that amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibody fragments of the present disclosure by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. A conservative amino acid substitution is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. Exemplary conservative amino acid substitutions are shown in Table 2.

[0059] In certain embodiments, the sequences described herein may have up to about 1, up to about 2, up to about 3, up to about 4, up to about 5, up to about 6, up to about 7, up to about 8, up to about 9, or up to about 10 amino acid residues modified and / or substituted.

[0060] Amino acids can be grouped according to common side chain properties as follows: TIFF2026035577000003.tif46170

[0061] In certain embodiments, non-conservative substitutions may involve exchanging a member of one of these classes for another class. Table 2 TIFF2026035577000004.tif255170

[0062] Other modified hinge regions of the present disclosure may be entirely synthetic and can be designed to possess desired properties, such as length, composition, and flexibility. For example, without limitation, modified hinge regions of the present disclosure can be altered to increase or decrease the flexibility of the hinge region. For example, without limitation, modifications that may increase the flexibility of a hinge region include, but are not limited to, substituting one or more amino acid residues with one or more amino acid residues that increase flexibility (e.g., glycine). In certain embodiments, modifications that may decrease the flexibility of a hinge region include, but are not limited to, substituting one or more amino acid residues with one or more amino acid residues that crease the rigidity of the polypeptide (e.g., proline).

[0063] B. Methods for Producing Antibody Fragments In certain embodiments, antibody fragments are produced by hinge engineering technology.

[0064] In certain embodiments, antibody fragment starting material for use in connection with the methods described herein can be obtained from any whole antibody (e.g., whole monoclonal antibody) using any suitable enzymatic cleavage and / or digestion technique, hi certain embodiments, antibody fragments can be obtained by cleavage with IdeS.

[0065] In certain embodiments, Fab molecules are produced by proteolytic digestion or recombinant expression. Proteolytic digestion was the original method for Fab production (6). Producing Fab molecules via proteolytic digestion results in the C-terminal sequence of the Fab heavy chain defined by a protease cleavage site. In turn, Fab molecules typically contain a portion of the upper hinge of an antibody. This upper hinge region of an antibody serves as a linker between the Fab and Fc regions but has no structural or functional role in the Fab molecule. It can be considered an unstructured appendage because it is often not fully resolved in the crystal structure of the Fab molecule (see Figure 1A). One therapeutic Fab molecule (abciximab, REOPRO®) directed against the platelet surface receptor GPIIb / IIIa is commercially produced by proteolytic cleavage with papain.

[0066] With advances in molecular cloning, recombinant expression of antibody fragments is an attractive route for generating Fab molecules (7). In contrast to proteolytic digestion as a production route, recombinant expression of Fab molecules offers flexibility in defining the length of the incorporated upper hinge region. In certain embodiments, Fab fragments are produced by recombinant expression.

[0067] The high affinity of antibodies is often achieved through bivalent target binding, which promotes avidity. In contrast, target binding of Fabs is monovalent. This often leads to lower target affinity compared to full-length IgG. By linking two Fab fragments to create F(ab')2, avidity can be restored while preserving key Fab characteristics, such as short serum half-life. In addition, targeting multiple disease mediators with specific antibodies has become increasingly important for the development of therapeutic antibodies (8). F(ab')2 molecules provide a natural scaffold for producing small bispecific antibody fragments. In contrast to the production of Fab molecules, recombinant expression of F(ab')2 is not naturally possible because the expressed Fab' molecules require non-native homo- or heterodimerization domains as fusion moieties (9, 10). Therefore, there are two main approaches to generating F(ab')2 molecules: (i) chemical conjugation and (ii) proteolytic digestion. In chemical conjugation, recombinantly expressed Fab' molecules are linked by homo- or heterobifunctional cross-linkers (3, 9, 11, 12). Similar to the proteolytic digestion approach for producing Fab molecules, several known proteases can cleave intact antibodies at the lower hinge region to produce F(ab')2 molecules (13). Pepsin is the most widely used proteolytic digestion, resulting in highly stable F(ab')2 molecules in which two Fab molecules are linked by two core-hinge disulfide bonds (14). More recently, the highly specific IgG-degrading enzyme IdeS from Streptococcus pyogenes has been described (15, 16). The use of IdeS allows for the generation of highly uniform products by eliminating the heterogeneity at the C-terminus observed with pepsin digestion (3). In certain embodiments, F(ab')2 fragments are produced by IdeS cleavage.

[0068] C. Recombinant Methods and Compositions Antibody fragments can be produced using recombinant methods and compositions described, for example, in U.S. Patent No. 4,816,567. In certain embodiments, an isolated nucleic acid encoding an antibody fragment described herein or a composition comprising such a nucleic acid is provided. Additionally, one or more vectors (e.g., expression vectors) comprising such a nucleic acid are provided. Host cells comprising such a nucleic acid are also provided. In certain embodiments, the host cell is a eukaryotic cell, e.g., a Chinese hamster ovary (CHO) cell or a lymphoid cell (e.g., a Y0, NS0, or Sp20 cell). In certain embodiments, a method of producing a Fab molecule is provided, the method comprising culturing a host cell comprising a nucleic acid encoding the Fab provided above under conditions suitable for expression of the Fab, and optionally recovering the Fab from the host cell (or host cell culture medium).

[0069] For recombinant production of a Fab, for example, nucleic acid encoding the Fab described above is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acid can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of binding specifically to genes encoding the heavy and light chains of the Fab).

[0070] Suitable host cells for cloning or expressing Fab-encoding vectors include the prokaryotic or eukaryotic cells described herein. For example, Fabs can be produced in bacteria. For expression of antibody fragments such as Fabs in bacteria, see, e.g., U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in E. coli.) After expression, Fabs can be isolated from the bacterial cell paste in a soluble fraction and further purified.

[0071] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for Fab-encoding vectors, including fungal and yeast strains whose glycosylation pathways have been "humanized." See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li et al., Nat. Biotech. 24:210-215 (2006).

[0072] Suitable host cells for the expression of glycosylated proteins can also be obtained from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for the transfection of Spodoptera frugiperda cells.

[0073] Plant cell cultures can also be used as hosts. See, e.g., U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES™ technology for producing antibodies in transgenic plants).

[0074] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to grow in suspension can be useful. Other examples of useful mammalian host cell lines include SV40-transformed monkey kidney CV1 line (COS-7), human embryonic kidney lines (e.g., 293 or 293 cells as described in Graham et al., J. Gen Virol. 36:59 (1977)), baby hamster kidney cells (BHK), mouse Sertoli cells (e.g., TM4 cells as described in Mather, Biol. Reprod. 23:243-251 (1980)), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), canine kidney cells (MDCK), buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), mouse mammary tumor (MMT 060562), e.g., Mather et al., Annals Other useful mammalian host cell lines include TRI cells, MRC 5 cells, and FS4 cells, as described in NYAcad. Sci. 383:44-68 (1982). - These include Chinese hamster ovary (CHO) cells, including CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)), and myeloma cell lines such as Y0, NS0, and Sp2 / 0. For a review of certain mammalian host cell lines suitable for Fab production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0075] D. Pharmaceutical Preparations Pharmaceutical formulations of the antibody fragments described herein, e.g., Fab and F(ab')2, are prepared by mixing the antibody having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)) in the form of a lyophilized formulation or aqueous solution. For example, but not limited to, lyophilized antibody formulations are described in U.S. Pat. No. 6,267,958. In certain embodiments, aqueous antibody formulations may include those described in U.S. Pat. No. 6,171,586 and WO 2006 / 044908, the latter formulations containing a histidine-acetate buffer.

[0076] In certain embodiments, antibody fragments of the present disclosure may be of greater than about 80%, greater than about 90%, greater than about 91%, greater than about 92%, greater than about 93%, greater than about 94%, greater than about 95%, greater than about 96%, greater than about 97%, greater than about 98%, greater than about 99%, greater than about 99.1%, greater than about 99.2%, greater than about 99.3%, greater than about 99.4%, greater than about 99.5%, greater than about 99.6%, greater than about 99.7%, greater than about 99.8%, or greater than about 99.9% purity.

[0077] Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citric acid, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as , serum albumin, gelatin, or immunoglobulin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include intercalating drug dispersants, such as soluble neutral active hyaluronidase glycoproteins (sHASEGPs), e.g., human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in U.S. Patent Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one embodiment, a sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases.

[0078] The formulations herein may also contain more than one active ingredient as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Such active ingredients are preferably present in combination in amounts that are effective for the purpose intended.

[0079] The compositions of the present disclosure can be administered by a variety of methods known in the art. The route and / or mode of administration varies depending on the desired results. The active compound can be prepared with a carrier that protects the compound from rapid release, such as a controlled-release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Many methods for preparing such formulations are described, for example, in "Sustained and Controlled Release Drug Delivery Systems," JR Robinson, ed., Marcel Dekker, Inc., New York, 1978. In certain embodiments, the pharmaceutical compositions are manufactured under the Good Manufacturing Practice (GMP) conditions of the U.S. Food and Drug Administration.

[0080] The carrier may be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, i.e., antibody fragment, may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

[0081] The active ingredient can be encapsulated in microcapsules prepared, for example, by coacervation techniques or interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and poly(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in microemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0082] Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules.

[0083] Formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, for example, by filtration through sterile filtration membranes.

[0084] The disclosed pharmaceutical compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the presence of microorganisms can be ensured both by the above-mentioned sterilization procedures and by the inclusion of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, etc. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, etc., in the compositions. Furthermore, prolonged absorption of the injectable dosage form can be brought about by the inclusion of agents that delay absorption, such as aluminum monostearate and gelatin.

[0085] In certain embodiments, when the antibodies of the present invention are administered to humans and animals as pharmaceuticals, they can be given alone or as a pharmaceutical composition containing, for example, about 0.01% to about 99.5% (or about 0.1% to about 90%) of the antibody fragment in combination with a pharmaceutically acceptable carrier.

[0086] E. Therapeutic Methods and Compositions Any of the antibody fragments provided herein can be used in methods of treatment. In certain embodiments, pharmaceuticals are provided for use as antibody fragments. In certain embodiments, antibody fragments are provided for use in treating specific disease indications. In certain embodiments, antibody fragments of the present disclosure can be used to treat ocular diseases and / or disorders. In certain embodiments, antibody fragments of the present disclosure can be used to treat diseases and / or disorders where the application of antibody fragments exhibiting short half-lives would be beneficial. In certain embodiments, antibody fragments are provided for use in methods of treatment.

[0087] In certain embodiments, the present disclosure provides an antibody fragment for use in a method for treating an individual having a particular disease, comprising administering to the individual an effective amount of the antibody fragment or a composition comprising the same. In certain embodiments, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described below. In certain embodiments, the present disclosure provides an antibody fragment for use in inhibiting a particular molecular pathway and / or mechanism. In certain embodiments, the present disclosure provides an antibody fragment for use in a method for inhibiting a particular molecular pathway and / or mechanism in an individual, comprising administering to the individual an antibody fragment effective to inhibit the particular molecular pathway and / or mechanism. In certain embodiments, the present disclosure provides an antibody fragment for use in a method for inhibiting a particular molecular pathway and / or mechanism in an individual, comprising administering to the individual an antibody fragment effective to activate the particular molecular pathway and / or mechanism. An "individual" according to any of the above embodiments may be a human.

[0088] In certain embodiments, the present disclosure provides use of an antibody fragment in the manufacture or preparation of a medicament. In certain embodiments, the medicament is for the treatment of a specific disease. In certain embodiments, the medicament is for use in a method for treating a specific disease, comprising administering an effective amount of the medicament to an individual having the disease. In certain embodiments, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described below. In certain embodiments, the medicament is for the inhibition or activation of a specific molecular pathway and / or mechanism. In certain embodiments, the medicament is for use in a method for inhibiting or activating a specific molecular pathway and / or mechanism in an individual, comprising administering to the individual an effective amount of the medicament to inhibit the specific molecular pathway and / or mechanism. An "individual" according to any of the above embodiments may be a human.

[0089] In certain embodiments, the present disclosure provides a method for treating a particular disease. In certain embodiments, the method comprises administering an effective amount of an antibody fragment to an individual having such a disease. In certain embodiments, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, for example, as described below. An "individual" according to any of the above embodiments may be a human.

[0090] In certain embodiments, the present disclosure provides methods for inhibiting a specific molecular pathway and / or mechanism in an individual. In certain embodiments, the methods comprise administering to the individual an effective amount of an antibody fragment to inhibit the specific molecular pathway and / or mechanism. In certain embodiments, the "individual" is a human.

[0091] In certain embodiments, the present disclosure provides pharmaceutical formulations comprising any of the antibody fragments provided herein, for example, for use in any of the above-mentioned therapeutic methods. In certain embodiments, the pharmaceutical formulations comprise any of the antibody fragments provided herein and a pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical formulations comprise any of the antibody fragments provided herein and at least one additional therapeutic agent, for example, as described below.

[0092] The antibody fragments of the present disclosure can be used in therapy either alone or in combination with other agents, for example, the antibody fragments of the present disclosure can be co-administered with at least one additional therapeutic agent.

[0093] Such combination therapy as described above encompasses combined administration (two or more therapeutic agents in the same or separate formulations) and separate administration, where administration of an antibody of the present disclosure can occur before, simultaneously with, and / or after administration of the additional therapeutic agent(s). In certain embodiments, administration of the antibody fragment and administration of the additional therapeutic agent occur within about one month, or within about one, two, or three weeks, or within about one, two, three, four, five, or six days of each other. The antibody fragments described herein can also be used in combination with radiation therapy.

[0094] The antibody fragment (and any additional therapeutic agents) may be administered by any suitable means, including parenteral, intrapulmonary, intraocular, and intranasal, as well as intralesional administration if desired for localized treatment. Parenteral infusions include intramuscular, intravenous, intraarterial, intraocular, intraperitoneal, or subcutaneous administration. Dosing may be by any suitable route, for example, by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is brief or chronic. Various dosing schedules are contemplated herein, including, but not limited to, single or multiple doses over various time points, bolus administration, and pulse infusion.

[0095] The antibody fragments are formulated, dosed, and administered in a manner consistent with good medical practice. Factors to consider in this regard include the particular disease being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disease, the site of drug delivery, the method of administration, the scheduling of administration, and other factors known to physicians. The antibody fragments are optionally, but not necessarily, formulated with one or more drugs currently used to prevent or treat the disease in question. The effective amount of such other drugs will depend on the amount of antibody fragment present in the formulation, the type of disease or treatment, and other factors discussed above. These will generally be used in the same dosages and by any route of administration as described herein, or about 1-99% of the dosages described herein, or at any dosage and by any route determined empirically / clinically appropriate.

[0096] For the prevention or treatment of disease, the appropriate dosage of the antibody fragment of the present disclosure (when used alone or in combination with one or more other additional therapeutic agents) depends on the type of disease being treated, the type of antibody fragment, the severity and course of the disease, whether the antibody fragment is being administered for prophylactic or therapeutic purposes, previous therapies, the patient's medical history, and response to the antibody fragment, as well as the discretion of the attending physician. The antibody fragment is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, an initial candidate dosage for administration to a patient may be about 1 μg / kg to 15 mg / kg (e.g., 0.1 mg / kg to 10 mg / kg), either by one or more separate administrations or by continuous infusion. A typical daily dose may range from about 1 μg / kg to 100 mg / kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer depending on the condition, treatment will generally be sustained until a desired suppression of disease symptoms occurs. One exemplary dosage of the antibody fragment would be in the range of about 0.05 mg / kg to about 10 mg / kg. Thus, one or more doses of about 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg, or 10 mg / kg (or any combination thereof) may be administered to the patient. Such doses may be administered intermittently, for example, weekly or once every three weeks (e.g., so that the patient receives about two to about twenty, or, for example, about six, doses of the antibody fragment). A higher initial loading dose, followed by one or more lower doses, may also be administered. The progress of this therapy is easily monitored by conventional techniques and assays.

[0097] It will be understood that any of the above formulations or methods of treatment can be carried out using immunoconjugates in place of, or in addition to, the antibody fragments of the present disclosure.

[0098] F. Immune complex The presently disclosed subject matter also provides immunoconjugates comprising an antibody fragment described herein conjugated to one or more cytotoxic agents, such as a chemotherapeutic agent or drug, a growth inhibitory agent, a protein, a peptide, a toxin (e.g., a protein toxin, an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioisotope. For example, an antibody fragment of the disclosed subject matter can be operatively linked (e.g., by chemical coupling, genetic fusion, noncovalent association, or otherwise) to one or more other binding molecules, such as another antibody, antibody fragment, peptide, or binding mimetic.

[0099] In certain embodiments, the immunoconjugate is an antibody-drug conjugate (ADC) in which an antibody fragment is conjugated to one or more drugs, including maytansinoids (see U.S. Pat. Nos. 5,208,020, 5,416,064, and EP 0425235 B1); auristatins, such as monomethyl auristatin drug moieties DE and DF (MMAE and MMAF) (U.S. Pat. No. 5,633,633); Nos. 5,483, 5,780,588, and 7,498,298); dolastatins: calicheamicin or derivatives thereof (see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296; Hinman et al., Cancer Res. 53:3336-3342 (1993), and Lode et al., Cancer Res. 58:2925-2928 (1998); anthracyclines such as daunomycin or doxorubicin (Kratz et al., Current Med. Chem. 13:477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem. Letters 16:358-362 (2006); Torgov et al., Bioconj. Chem. 16:717-721 (2005); Nagy et al., Proc. Natl. Acad. Sci. USA 97:829-834 (2000); Dubowchik et al., Bioorg. & Med. Chem. Letters 12:1529-1532 (2002), King et al., J. Med. Chem. 45:4336-4343 (2002), and U.S. Patent No. 6,630,579); methotrexate; taxanes such as vindesine, docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; trichothecines; and CC1065.

[0100] In certain embodiments, the immunoconjugate comprises an antibody fragment described herein conjugated to an enzymatically active toxin or fragment thereof, including, but not limited to, diphtheria A chain, a non-binding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and a trichothecene.

[0101] In certain embodiments, the immunoconjugate comprises an antibody fragment as described herein conjugated to a radioactive atom to form a radioconjugate. A variety of radioisotopes are available for the production of radioconjugates. Non-limiting examples include At 211 , I 131 , I 125 , Y 90 ,Re 186 ,Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 and radioactive isotopes of Lu. When a radioconjugate is used for detection, it can be a radioactive atom, such as tc99m or I, for scintigraphy studies. 123 , or spin labels for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, mri), such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron.

[0102] Conjugates of antibody fragments and cytotoxic agents can be made using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azido compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionucleotides to antibodies. See WO 94 / 11026. The linker can be a "cleavable linker" that facilitates release of the cytotoxic drug inside the cell. For example, an acid-labile linker, peptidase-sensitive linker, photolabile linker, dimethyl linker, or disulfide-containing linker (Chari et al., Cancer Res. 52:127-131 (1992); U.S. Pat. No. 5,208,020) may be used. Non-limiting examples of linkers are disclosed above.

[0103] The immunoconjugates herein expressly contemplate, but are not limited to, such conjugates prepared with crosslinker reagents including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate), which are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, IL, USA).

[0104] G.Product In certain embodiments of the present disclosure, an article of manufacture containing materials useful for the treatment, prevention, and / or diagnosis of the aforementioned disorders is provided. The article of manufacture includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The container can be formed from a variety of materials, such as glass or plastic. The container holds a composition that, by itself or in combination with another composition, is effective for the treatment, prevention, and / or diagnosis of a condition and can have a sterile access port (e.g., the container can be an IV solution bag or a vial with a stopper pierceable by a hypodermic needle). At least one active agent in the composition is an antibody fragment of the present disclosure. The label or package insert indicates that the composition is used to treat the selected condition. Furthermore, the article of manufacture can include (a) a first container containing a composition comprising the antibody fragment of the present disclosure, and (b) a second container containing a composition comprising an additional cytotoxic or therapeutic agent. The article of manufacture of this embodiment of the present disclosure may further include a package insert indicating that the composition can be used to treat a particular condition. Alternatively, or additionally, the article of manufacture may further include a second (or third) container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0105] It will be understood that any of the above products may contain immunoconjugates instead of, or in addition to, the antibody fragments described herein. [Example]

[0106] The following are examples of the methods and compositions of the present disclosure. Given the summary provided above, it will be understood that various other embodiments may be practiced.

[0107] Example 1 - Avoidance of pre-existing anti-hinge antibody responses by hinge engineering Fab and F(ab')2 antibody fragments serve as alternative formats to full-length antibodies in therapeutic and immunoassays. They offer the advantages of small size, short serum half-life, and lack of effector function. Several proteases associated with invasive diseases are known to cleave antibodies at the hinge region, resulting in anti-hinge antibodies (AHA) directed against neoepitopes. Pre-existing AHA in serum can act as a surrogate Fc, reintroducing the Fc properties missing in antibody fragments. While this response is desirable during the natural process of fighting disease, it is generally unnecessary for therapeutic antibody fragments. In this study, we identified a cleavage site in the lower hinge region of an antibody that maintains efficient proteolytic cleavage by IdeS protease. The resulting neoepitopes at the C-terminus of F(ab')2 fragments lack detectable pre-existing AHA, providing a practical route for in vitro F(ab')2 production by proteolytic digestion when a pre-existing AHA response is undesirable. In this study, the upper hinge region of the antibody was also investigated, providing a detailed analysis of the contribution of the C-terminal residues of the upper hinges of human IgG1, IgG2, and IgG4 to pre-existing AHA reactivity in human serum. No pre-existing antibodies were observed against Fabs of IgG2 and IgG4 isotypes, but significant responses were observed against most residues in the upper hinge of human IgG1. 225 L mutation (herein referred to as “T 225 L variant) and the native C-terminal D 221 identified as a solution with minimal seroreactivity. This work enabled the production of Fab and F(ab')2 fragments for therapeutic and immunoassays with minimal reactivity to existing AHAs.

[0108] Materials and Methods Plasmid construction and antibody expression: Antibodies were cloned into E. coli expression vectors (9, 23) or mammalian expression vectors (24) by standard molecular biology techniques as previously described. E. coli expression was performed as described by Simmons et al. (23). IgG and Fab were expressed in 30 mL transient transfection cultures of CHO (25) or HEK293T (26) cells as previously described.

[0109] Cloning, Expression, and Purification of IdeS: IdeS was expressed as an N-terminal glutathione S-transferase (GST) fusion protein. The mature sequence of IdeS from Streptococcus pyogenes MGAS1882 (Uniprot ID H8HDR0) was codon-optimized for E. coli expression, synthesized by GeneArt™, and cloned into an E. coli expression vector (23) using standard molecular biology techniques. IdeS was expressed using the conditions described by Simmons et al. (23) and purified using a glutathione Sepharose (GSH) column. The elution fraction from the GSH column in 50 mM Tris-HCl (pH 8.0), 20 mM glutathione was concentrated, loaded onto an S200 column, and eluted with 200 mM KHPO (pH 6.2), 250 mM KCl.

[0110] Antibody and Fab purification: After expression, cells were pelleted by gravity. The supernatant was transferred to a 50 ml Falcon tube (Corning, Corning, NY, USA). 400 μl of 50% MabSelect SuRe™ protein affinity slurry or Gamma Bind™ Plus slurry (GE Healthcare, Pittsburgh, PA, USA) was added to the supernatant for IgG and Fab purification, respectively. The mixture was incubated overnight at room temperature on an Innova 2000 platform shaker (New Brunswick Scientific, Enfield, CT, USA). The supernatant was removed, and the resin was transferred to a 96-well, 2 ml filter plate with 25 μm membranes (Thompson Instrument, Oceanside, CA, USA). The resin was washed three times with 1 ml of 1x PBS (pH 7.4) by centrifugation at 1,120x g for 5 minutes using a Sorvall™ HT6 centrifuge (Thermo Scientific, Waltham, MA, USA). For Fab purification, the resin was further washed with 0.2x PBS pH 5.0 before elution. IgG was eluted using 50 mM phosphate pH 2.9, and the eluate was neutralized with 20x PBS pH 11.0 by centrifugation at 1,000x g for 5 minutes. Fab fragments were eluted using 10 mM sodium citrate pH 2.9 and neutralized with 0.3 M Tris pH 9.0. The eluted IgG and Fab were filtered through a 0.2 μm 96-well filter plate (Orochem, Naperville, IL, USA) by centrifugation at 1,000×g for 5 min using a Sorvall™ HT6 centrifuge (Thermo Scientific, Waltham, MA, USA).

[0111] IdeS digestion of IgG hinge mutants: 1 mg / ml IgG was incubated with a defined IdeS:IgG ratio (w / w) for 24 hours at 37° C. For scaled-up digestions to generate large amounts of highly pure F(ab')2 material for AHA assays, IdeS:IgG ratios of up to 1:10 were used to promote complete digestion.

[0112] F(ab')2 purification after IdeS IgG cleavage: The IdeS-cleaved sample was diluted with 25 mM sodium acetate (pH 4.4) (Buffer A) and loaded onto a 1 mL SP Sepharose high-performance strong cation exchange column (GE Healthcare, Pittsburgh, PA, USA) equilibrated in Buffer A at 150 cm / h (0.7 cm diameter, 10 cm bed height). The column was washed to baseline with Buffer A, and F(ab')2 was eluted with a linear salt gradient from 0 to 0.5 M NaCl over 30 column volumes. The eluate was neutralized to pH 7.0 by adding 3 M Tris (pH 9.0) and filtered through a 0.22 μm STERIFLIP® filter (EMD Millipore, Billerica, MA, USA). The SP-eluted F(ab')2 was further purified on a MonoS 5 / 50 GL strong cation exchange column (GE Healthcare, Pittsburgh, PA, USA) after dilution with Buffer A to reduce the conductivity to <5 mS / cm. The column was washed with Buffer A to baseline (<0.05 mAU), and the F(ab')2 was eluted with a salt gradient of 0 to 0.6 M NaCl over 40 column volumes. The eluted F(ab')2 solution was neutralized with 3 M Tris (pH 9.0) to adjust the pH to 7.0 and filtered through a 0.22 μm STERIFLIP® (EMD Millipore, Billerica, MA, USA).

[0113] Mass spectrometry of Fab and F(ab')2 fragments: Mass spectrometry data were acquired using an Agilent 6224 TOF LC-MS system (Agilent Technology, Santa Clara, CA, USA). F(ab')2 was reduced with 100 mM dithiothreitol at 37°C for 20 minutes. Polypeptide chains were separated using a PLRP-S reversed-phase column (Agilent Technologies, Santa Clara, CA, USA). The intact masses of the reduced light and heavy chains were obtained by Maximum Entropy Deconvolution using MassHunter software (Qualitative Analysis B.03.01).

[0114] Protein analysis by capillary electrophoresis: All samples were prepared by mixing 5 μl of sample volume with 7 μl of HT protein expression sample buffer and incubated at 70°C for 5 minutes. 32 μl of water was added to the sample and centrifuged at 1,000×g for 5 minutes. The chip was prepared according to the manufacturer's instructions provided in the LabChip GXII user guide, and samples were analyzed using a Caliper GX II microfluidic system (PerkinElmer® Biotechnology, Waltham, MA, USA). All reagents were obtained from PerkinElmer®.

[0115] Existing anti-hinge antibody enzyme-linked immunosorbent assay (ELISA): MAXISORP® plates (384-well, Nunc, Thermo Fisher Scientific, Rochester, NY, USA) were coated with 1 μg / ml F(ab')2 or Fab in 50 mM carbonate (pH 9.6) overnight at 4°C. The plates were washed with 0.05% polysorbate 20 in PBS (pH 7.4) and then blocked with 0.5% BSA, 15 ppm Proclin in PBS (pH 7.4). Pooled human sera from 25 female and 25 male individuals (Bioreclamation IVT, Westbury, NY, USA) were serially diluted in assay buffer (0.5% BSA, 0.05% polysorbate 20, 15 ppm Proclin™ in PBS (pH 7.4)) and added to the plates. After a 2-hour incubation, bound pre-existing anti-hinge antibodies were detected using horseradish peroxidase (HRP) goat anti-F(ab')2-conjugated anti-human IgG Fc (Jackson ImmunoResearch, West Grove, PA) in assay buffer, followed by 3,3',5,5'-tetramethylbenzidine (TMB, Moss Inc., Pasadena, MD, USA) as substrate. The reaction was stopped with 1 M phosphoric acid, and absorbance was read at 450 nm. Absorbance readings at a 1:30 dilution were used in the figures to allow for representation of all samples. Comparative results were observed at a 1:10 serum dilution. To calculate relative AHA reactivity, F(ab')2 titer curves were fitted using a four-parameter curve-fitting program (KaleidaGraph, Synerg Software, Reading, PA). The median OD (average OD of the top and bottom OD readings) of the F(ab')2 titer curve was determined. The dilution of Fab DKTHT (SEQ ID NO: 21) and F(ab')2 corresponding to this median OD was calculated and used to calculate relative AHA reactivity.

[0116] SDS-PAGE and immunoblotting: For SDS-PAGE, 5 μg of purified F(ab')2 variants and GST-IdeS were mixed with SDS-sample buffer, heated at 95°C for 5 min, and spun at 16,000 relative centrifugal force for 1 min. Samples were loaded onto NuPAGE 4-12% BisTris / MES gels (Invitrogen). For immunoblotting, 5 ng of protein sample was used for SDS-PAGE. The gel was transferred to a nitrocellulose membrane by IBLOT® (Invitrogen) and immunoblotted with anti-IdeS (Genovis, USA, catalog no. A3-AF1-010, lot no. A3AF1-7C17H) as the primary antibody and IRDye800CW-conjugated donkey anti-goat antibody (Li-COR®, USA, catalog no. 926-32214, lot no. B80821-03) as the secondary antibody, and imaged using a LI-COR® Odyssey® Imager (LI-COR®, USA). Odyssey® two-color protein molecular weight markers (LI-COR®, USA) were used for immunoblotting, and pre-stained SEEBLUE® Plus2 (Invitrogen, USA) was used for Coomassie-stained gels.

[0117] Protein stability measurement by differential scanning fluorimetry: Protein stability was determined on a Biorad CFX96 TOUCH™ Real-Time System (Biorad, USA) at a final dilution of 1:200 in SYPRO® Orange dye stock (Molecular Probes™, USA). 1 μl of SYPRO® Orange dye stock was added to 24 μl of purified antibody at 100 μg / ml. Fluorescence of the final 25 μl sample in PBS was recorded from 20 to 100°C (0.2°C increments, 10-second hold between steps).

[0118] result The Fab C-terminus determines the response to pre-existing AHAs: Originally, the response of autoantibodies in human serum to the upper hinge of the Fab molecule was studied using the papain-cleaved antibody abciximab (5). Papain cleavage results in the C-terminal H 224Later, a more comprehensive study was performed using biotinylated peptide analogs to dissect the contribution of individual C-terminal residues of the upper hinge (20). In this study, the upper hinge residue K 222 ~H 224 Only minimal AHA reactivity was observed with T as the C-terminal residue. 225 No signal was observed for the peptide with the upper hinge residue D. 221 ~T 225 The Fab-tail (Figure 1A) spanning 100 s may confound the results because it is presented outside the context of the intact molecule. Therefore, we investigated the contribution of the Fab-tail to binding to pre-existing AHA in the setting of intact Fab.

[0119] Recombinant expression of Fab molecules in E. coli and mammalian cells allows for the facile production of molecules with defined C-termini without the need for proteolytic cleavage. To ensure the integrity of the C-terminus, the exact mass of purified Fab was confirmed by intact mass spectrometry. Fab molecules were coated onto microtiter plates, and after incubation with pooled human donor serum, binding of pre-existing AHA was quantified by anti-Fc detection. Consistent with previous studies (20), the C-terminal T 223 (having the sequence DKT, also referred to herein as "CDKT" (SEQ ID NO: 17)) showed the highest reactivity to existing AHAs among all upper hinge mutants (Figure 1B). A striking difference from previous studies is the C-terminal T 225 (having the sequence DKTHT (SEQ ID NO: 21), also referred to herein as "CDKTHT" (SEQ ID NO: 14). This variant did not bind to AHA as a peptide (20), but substantial AHA reactivity was observed when tested as a Fab. 221 In the case of Fab (having sequence D, also referred to herein as "CD"), binding of AHA was reduced to almost background. 221 Terminating the α provides a solution to minimize recognition by existing AHAs while maintaining the native antibody sequence.

[0120] As demonstrated by these experiments, the C-terminal Fab residues have a profound effect on AHA binding. Binding by existing antibodies can be prevented by even a single amino acid change at the C-terminus, allowing for the use of D3 to minimize reactivity to AHA. 221 To place a non-natural residue at the C-terminus of the Fab, we investigated whether T 225 The L mutant was introduced and tested for AHA binding. 225 The L mutant has been described previously (7). The mutation disrupted existing AHA binding (Figure 1B), further highlighting the importance of the C-terminus in binding. 225 To rule out the possibility that the reduced AHA in L was due to reduced coating efficiency, a similar fold reduction in AHA signal was again observed when the Fab molecules were captured using an antigen capture format. To determine whether this observation could be generalized, three different Fabs were incubated with pooled human serum, and binding of pre-existing antibodies was detected by ELISA. Significant signals were observed for the three different Fabs with the DKTHT (SEQ ID NO: 21) C-terminus, while binding of the pre-existing antibodies, D 221 and T 225 Reduced binding to the LC terminus was indeed detected (Fig. 1C).

[0121] Next, we investigated Fab molecules of the IgG2 and IgG4 isotypes. Although IgG1, IgG2, and IgG4 are commonly used therapeutic antibodies, the use of IgG2 and IgG4 Fabs has not been exploited for therapeutic drug development to date. Therefore, we investigated the use of IgG2 and IgG4 Fabs with intact upper hinge regions (Figure 1D, C-terminal K, respectively). 218 and P 225 ) were tested. In contrast to IgG1 Fab, IgG2 and IgG4 Fab were not recognized by the existing AHA. Next, the upper hinge of IgG4 was cleaved. The length of the upper hinge of the IgG4 isotype is shorter than that of IgG1 (see Figures 6A-B), but the cysteine ​​involved in the heavy-light chain inter-disulfide is C.H 1Because it is located at the center of the primary structure, residue K 218 and Y 219 These truncated upper hinge Fabs showed a signal similar to that of the intact IgG1 upper hinge (Figure 1B).

[0122] All Fab molecules within the same isotype produced similar expression levels in E. coli and CHO (Figure 7). No changes in thermal stability were observed within the same isotype (Table 3). The thermal stability of IgG2 and IgG4 Fabs was reduced by approximately 6°C compared to the IgG1 isotype.

[0123] In conclusion, there are several Fab formats that have minimal reactivity to existing AHAs: IgG1-D 221 , IgG1-T 225 L, IgG2 and IgG4. Table 3. Thermal stability of T30M Fab determined by differential scanning fluorimetry. TIFF2026035577000005.tif80170

[0124] IgG1 with the lower hinge of IgG2 cannot be efficiently cleaved: existing AHAs directed against the lower hinge region of F(ab')2 have been extensively described in the literature (13, 27). Similar to AHAs directed against the upper hinge of Fab molecules, these AHAs may act as surrogate Fc, thus introducing assay artifacts. Therefore, the development of a F(ab')2 format that prevents AHA binding is desirable. While AHAs in serum have been found to be directed against F(ab')2 of IgG1 isotypes, it has not been possible to establish the presence of autoantibodies directed against the lower hinge of IgG2 isotypes. Interestingly, the lack of such autoantibodies is consistent with the inability of physiologically relevant human proteases to efficiently cleave IgG2 into F(ab')2 fragments (28). However, inefficient cleavage of IgG2 was observed using the IdeS protease (28). IdeS is a G 236IdeS is an IgG-specific endoprotease derived from Streptococcus pyrogenes that cleaves after the F(ab')2 region. In addition to the cleavage site in the antibody hinge region, it recognizes a second site in the Fc region, which contributes to its high specificity for IgG (15, 16). Inefficient cleavage of IgG2 antibodies may be caused by inactive sites outside the cleavage site. Therefore, the lower hinge residues of IgG2 were grafted onto IgG1 to create an IgG1-2 chimera (Figure 2B). The cleavage efficiency at different IdeS:IgG ratios was tested (Figure 2C). While wild-type IgG1 was efficiently cleaved to F(ab')2 at an IdeS:IgG ratio of 1:500, at least a 50-fold higher protease concentration was required to achieve similar cleavage of the IgG1-2 chimera. It was concluded that sequence differences in the lower hinge region at least partially contribute to the poor cleavage efficiency of IgG2 antibodies. Therefore, IgG1-2 chimeras may not be a practical strategy for generating F(ab')2 molecules that do not bind to existing AHAs.

[0125] Characterization of the P1 and P2 positions for efficient IdeS cleavage: In the described Fab experiments, pre-existing AHA binding was observed in a single C-terminal T 225 It was demonstrated that this could be prevented by L mutation. A similar strategy was used for F(ab')2. As a first step, residues at the P1 (EU236) and P2 (EU235) sites that allow cleavage by the IdeS protease were identified. The L at the P2 position for IdeS was 235 , L 235 V, L 235 I or L 235A set of 76 Fab variants containing M was generated and combined with any amino acid except cysteine ​​at P1 (Figures 3D and 3E). The antibodies were purified and digested with IdeS at an IdeS:IgG ratio of 1:10 to identify variants that could be cleaved by IdeS. This high protease-to-antibody ratio was chosen to evaluate proteolysis without taking cleavage efficiency into account. Seven variants that were cleaved by IdeS were identified (Figure 3A). While the P2 position tolerated all four residues tested, only two amino acids with very small side chains, namely, natural glycine and alanine, were accepted at the P1 position. This subset was further investigated for cleavage efficiency at three different IdeS:IgG ratios: 1:500, 1:200, and 1:100 (Figure 3B) and 1:10 (Figure 3C). The variant L at the P2 position was also identified (Figure 3D). 235 V demonstrated minimal change in cleavage efficiency compared to the wild-type sequence. All other mutants were characterized by a predominantly single cleavage on only one side of the hinge, leaving the other side of the antibody intact. L with a glycine at position P1 235 I and L 235 The M variant was completely cleavable at an IdeS:IgG ratio of 1:200, whereas all P2 variants with an alanine at P1 required significantly higher amounts of protease. Thus, efficient cleavage by IdeS requires a glycine at the P1 position.

[0126] The C-terminal F(ab')2 variants do not prevent recognition by pre-existing AHAs: IdeS can confound the results of pre-existing AHA assays with false-positive results. This may be explained by binding of serum antibodies by IdeS and their subsequent recognition by anti-Fc detection antibodies. Therefore, we ensured that the F(ab')2 molecules used in the assay did not contain IdeS from a previous proteolytic reaction. Removal of IdeS in the purified F(ab')2 protein was confirmed by SDS-PAGE followed by Coomassie staining and anti-IdeS immunoblotting (Figure 4A).

[0127] The purified P1 and P2 F(ab')2 variants were then tested for their recognition by pre-existing AHAs (Figure 4B). Although the signal was reduced compared to wild-type, none of the antibodies with C-terminal modifications eliminated reactivity with serum autoantibodies. Changes to the P2 position had only a moderate effect. The G at the P1 position 236 The A change reduced the signal to levels comparable to that of Fab. Thus, without being limited to a particular theory, it is not possible to design a lower hinge with amino acid mutations that retain cleavage by IdeS while simultaneously eliminating pre-existing antibody responses to F(ab')2.

[0128] Deletions in the lower hinge block recognition by existing AHA while preserving IdeS cleavage: We used an alternative strategy to remove the AHA epitope by truncating the lower hinge. We left the P1 (EU236) and P2 (EU235) residues intact due to their importance in cleavage efficiency, and generated single and double residue deletions at the P3 (EU234), P4 (EU233), and P5 (EU232) sites of IdeS to identify residues with minimal impact on cleavage efficiency (see Figure 5F). Significantly lower cleavage efficiency was observed with the deletion of the P3 position; therefore, this position was not considered in further studies (Figure 5A). Antibodies with deletions of P4, P5, and a combination of both (ΔP45, also referred to herein as deletion of amino acid residues at EU232-233) were tested for binding to AHA (Figure 5B). Lower signals were observed with these mutants compared to the wild-type hinge sequence. To further reduce hinge recognition by pre-existing AHA, the deletion was extended to include residues P6 (EU231) and P7 (EU230) (see Figure 5F). Deletion of the P4-P7 sites (ΔP4567, also referred to herein as deletion of the amino acid residues at EU230-233) resulted in a moderately reduced cleavage efficiency, while deletion of the P4-P6 sites (ΔP456, also referred to herein as deletion of the amino acid residues at EU231-233) had cleavage efficiency equivalent to that of the wild-type at an IdeS:IgG ratio of 1:200 (Figures 5C and 8). Neither variant resulted in pre-existing AHA recognition (Figure 5D). To ensure that the high cleavage specificity of IdeS was maintained, F(ab')2 cleaved by IdeS was analyzed using ESI-TOF mass spectrometry. 236 A single mass corresponding to the predicted cleavage site was observed for wild-type F(ab')2 as well as the ΔP456 mutant F(ab')2 (Fig. 5E).

[0129] As shown in Figure 11, the observed AHA binding signal of the ΔP456 mutant F(ab')2 was significantly higher than that of the two Fab C-terminal mutants, i.e., Fab D 221 and Fab T 225The dilution corresponding to OD (1.15) at the center of the F(ab')2 titer curve was 70 and 14 for F(ab')2 and Fab, respectively. Without being limited to a particular theory, the Fab T observed with F(ab')2 is 225 The five-fold higher AHA activity compared to F(ab')2ΔP456 and Fab T can be explained by the possibility of bivalent binding of AHA to F(ab')2, indicating that the F(ab')2 molecule must avoid pre-existing AHA. 225 To rule out the possibility that the reduced AHA reactivity seen with L was due to reduced coating efficiency, AHA was detected using an antigen capture format. OD readings (n=4) were obtained for F(ab')2, ΔP456 F(ab')2, Fab, and Fab T at a 1:30 serum dilution. 225 For the antigen-coated wells that received L and buffer, the values ​​were 2.2±0.1, 0.34±0.03, 1.3±0.1, 0.36±0.02, and 0.32±0.01, respectively. Therefore, these results suggest that the ΔP456 F(ab')2 and Fab T 225 It was confirmed that L had reduced AHA reactivity compared to their corresponding wild-type molecules.

[0130] These data indicate that the ΔP456 variant offers a solution to circumvent the pre-existing AHA response to the lower hinge of F(ab')2, while maintaining the possibility of producing F(ab')2 antibody fragments by the well-established pathway of proteolytic digestion.

[0131] Deletions in the lower hinge of C-terminal F(ab')2 mutants: To determine whether recognition by existing AHAs is altered, C-terminal mutants with deletions in the lower hinge region were analyzed. Alanine or glycine at P1, along with L at P2 for IdeS, were used. 235 , L 235 V, L 235 I or L 235A set of six Fab variants containing M was generated with various hinge region deletions (Figure 9). This subset was further investigated for cleavage efficiency at three different IdeS:IgG ratios: 1:10 (Figure 9A), 1:100 (Figure 9B), and 1:500 (Figure 9C). As shown in Figure 9D, mutant L combined with the deletion at P5 235 V or G 236 As shown in Figure 10, deletion of L at P2 together with alanine or glycine at P1 resulted in a reduced AHA signal compared to deletion of P5 alone. 235 V, L 235 I or L 235 The ΔP456 mutant containing M showed a similar reduction in AHA signal compared to ΔP456 alone (Fig. 10B), but the ΔP456 deletion alone showed better cleavage efficiency compared to the mutant combined with ΔP456 (Fig. 10A).

[0132] Consideration Antibody fragments, such as Fab and F(ab')2, are attractive therapeutic formats when short systemic half-lives and effector-silent molecules are simultaneously desired. Certain fragments are also natural products of proteases associated with invasive diseases such as tumor cells and bacteria, and are generated in an attempt to evade immune surveillance. As a result, the C-terminal neo-epitopes of Fab and F(ab')2 fragments result in AHAs that can be recognized by the immune system and provide surrogate Fc.

[0133] In this study, we investigated the reactivity of pre-existing AHAs against individual C-terminal residues spanning the upper hinge region of human IgG1, IgG2, and IgG4 isotypes. While it was previously reported that there are no pre-existing AHAs against neoepitopes in the lower hinge of IdeS-cleaved human IgG2 antibodies, reactivity against the upper hinge of human isotypes has been incompletely investigated. In this study, pre-existing AHAs against the upper hinge of IgG2 and IgG4 isotypes were not detected. This, in turn, may suggest that these isotypes are not targets of proteases in invasive diseases. This may be explained by the effector-attenuated nature of these isotypes and the fact that removal of the Fc region of these isotypes does not provide an advantage to tumors and bacteria. In contrast, the IgG1 isotype appears to be the primary target of these proteases. Indeed, several proteases, including plasmin, human neutrophil elastase, and LysC, have been described to cleave in the upper hinge of human IgG1. This study is incompletely documented. 221 This indicates that there are existing AHAs for all cleavage sites in the upper hinge of human IgG1 except for D. 221 The absence of AHA at the C-terminal T may reflect the inability of human proteases to cleave after this residue or the inability to generate antibodies against this neoepitope. 223 The highest reactivity was observed to the Fab. Interestingly, this C-terminus coincides with the cleavage point of human neutrophil elastase, a protease secreted by neutrophils and macrophages during inflammation to destroy bacteria and host tissue (29).

[0134] Existing AHAs can rapidly recruit effector function to molecules that are engineered to lack effectors. The use of Fabs of the IgG2 or IgG4 isotype provides one strategy for delivering molecules without a pre-existing antibody response. Alternatively, the heavy chain C-terminus can be modified with a T 225 Introducing non-native residues such as L mutations or converting the upper hinge to D221 Cleavage at T is the strategy for the IgG1 isotype. 225 The L mutation eliminates responses to pre-existing AHAs, implying that, in principle, it could also elicit an immune response. This is further supported by recent studies on anti-TNFR1 domain antibodies (34). During in vitro screening, pre-existing human anti-V H Although the addition of the C-terminal alanine was sufficient to reduce antibody binding, one subject was found to develop high levels of antibodies specific to the modified C-terminus in a Phase I clinical trial. In addition, potential exopeptidase activity on the longer tail may eventually result in neoepitopes recognized by existing AHAs. Fab-D 221 Complete removal of the unstructured upper hinge, as in (30), further minimizes the risk of such secondary responses. This is supported by the crystal structure of an anti-hinge antibody crystallized in complex with a peptide spanning the IdeS-cleaved lower hinge of human IgG1. (30) This peptide is bound to the antibody in an extended conformation, suggesting that cleavage of either the upper or lower hinge can successfully remove neoepitopes and suppress antibody immune responses against the hinge region.

[0135] These findings may also have implications for the design of studies in cynomolgus monkeys. While the lower hinge region is highly conserved between cynomolgus monkeys and human IgG, there are substantial differences in the upper hinge region (31), which may prevent cross-species reactivity. This may have implications for toxicological studies in cynomolgus monkeys, as the disadvantages from existing AHAs relative to human Fabs cannot be addressed by these studies.

[0136] Beyond therapeutic uses, Fab-D 221 This example also demonstrates that the upper hinge is generally not resolved in crystal structures due to its unstructured nature, which can be considered for recombinant expression of Fabs for crystallographic studies. 221This demonstrates that this can be achieved with a structure. Eliminating unstructured regions can further improve crystallization outcomes.

[0137] To date, the most efficient route for generating F(ab')2 molecules is via proteolytic digestion, with highly specific proteases such as IdeS being preferred. As previously discussed, pre-existing AHAs are present on the lower hinge of F(ab')2 molecules. AHA titers are higher for IdeS-cleaved antibodies compared to Fabs. Without being limited to a particular theory, this may be due to the bivalent nature of F(ab')2, which provides the binding element for avidity in this assay, or the higher initial abundance of F(ab')2, leading to increased titers. To remove AHA reactivity while maintaining IdeS cleavage efficiency, several strategies were employed.

[0138] Because C-terminal residues play a critical role in epitope recognition (22), the first strategy was to mutate the C-terminal residues of F(ab')2 to eliminate AHA binding activity. However, this was not possible due to the strict requirement of glycine at the P1 position for efficient cleavage by IdeS. A selected set of mutations at the P2 position had only a moderate effect on AHA binding, further confirming the importance of the C-terminal residues for reactivity with AHA. The concordant differences in AHA binding and IdeS cleavage efficiency of the P1 and P2 mutants likely explain why IgG2 isotypes with valine at P2 and alanine at P1 are less susceptible to anti-hinge antibody responses. The requirement of glycine at the P1 position for efficient cleavage is accompanied by the high conservation of this residue within different isotypes and across multiple species.

[0139] By deleting three residues in the lower hinge (ΔP456), we were able to maintain the cleavage efficiency of IdeS while eliminating pre-existing AHA recognition. This demonstrates that the location upstream of the P3 site plays a minor role in efficient cleavage. In addition to eliminating pre-existing AHA reactivity, truncating the lower hinge may attenuate the immune response to this epitope. Based on structural studies, AHA binds to the lower hinge in an extended conformation, and the five C-terminal residues interact with the antibody's complementarity-determining region (CDR) (30). By removing three residues from the lower hinge, only four residues remained after IdeS cleavage. This short sequence may be insufficient for a robust immune response and may reduce the likelihood of de novo antibody development against the engineered hinge.

[0140] Existing antibodies have also been demonstrated to have reduced effector function in vitro (32), which can also confound immunogenicity assays during drug development. The majority of antitherapeutic antibodies (ATAs) against humanized antibodies target the idiotype rheumatoid factor, although low-affinity antibodies directed against the Fc region have been described. One way to eliminate artifacts due to rheumatoid factor in immunogenicity assays is to use antibody fragments lacking the Fc region. However, it is important to use fragments that are not recognized by other existing antibodies. These findings provide several Fab and F(ab')2 formats that can meet these criteria.

[0141] In summary, by selecting an appropriate antibody fragment, it is possible to avoid recognition by existing AHAs. Regarding the Fab molecule, several options exist: (1) use of IgG2 or IgG4 isotypes; (2) selection of the C-terminal residue (T 225 L) mutation, or (3) mutation of Fab to residue D 221The F(ab')2 molecule terminates at the cleavage site. Options are more limited for the F(ab')2 molecule due to the need for proteolytic digestion. However, deletions in the lower hinge of IgG1 have been identified that maintain high cleavage efficiency and specificity and eliminate reactivity with existing AHAs. Use of these formats may further minimize safety concerns associated with antibody fragments in therapeutic settings and remove bottlenecks in assay development.

[0142] Example 2 - F(ab')2ΔP456 has reduced AHA-mediated FcγRIIIa and C1q binding It has previously been described that purified AHA antibodies can act as surrogate Fc to restore ADCC / CDC function lost by IdeS-generated F(ab')2 (20, 22). To investigate whether the reduced binding of AHA by the disclosed engineered Fab and F(ab')2 variants is further reflected by reduced recruitment of Fcγ receptors and C1q, cross-linking experiments were used. To assess AHA binding to FcγRIIIa, human serum was added to Fab- or F(ab')2-coated wells as described above and incubated for 2 hours. After washing the plates, soluble FcγRIIIa(V158)-His-GST (consisting of the extracellular domain fused at the carboxy terminus to Gly-His6-glutathione-S-transferase ("Gly-His6" as disclosed in SEQ ID NO: 28)) was added at 0.5 μg / ml. Bound FcγRIIIa(V158)-His-GST was detected using horseradish peroxidase-conjugated mouse anti-His antibody (Penta-His (SEQ ID NO: 29), Qiagen, Germantown, MD) followed by TMB as substrate. To assess AHA binding to human C1q, human serum was added to Fab- or F(ab')2-coated wells as described above and incubated for 2 hours. After washing the plates, purified human C1q (Quidel, San Diego, CA) was added. Bound C1q was detected with goat anti-C1q antibody (Nordic Immunological Laboratories, Tilburg, The Netherlands) followed by rabbit anti-goat IgG-HRP (Jackson ImmunoResearch, West Grove, PA) and TMB as substrate.

[0143] Significant binding of FcγRIIIa and C1q to F(ab')2 was observed, while almost no signal was detected for the ΔP456 F(ab')2 variant, indicating that engineering the F(ab')2 variant significantly reduced the risk of ADCC / CDC activation. OD readings (n=3) were 0.45±0.05, 0.10±0.02, and 0.09±0.02 for FcγRIIIa binding, and 0.98±0.09, 0.158±0.004, and 0.107±0.009 for C1q binding, respectively, for F(ab')2, ΔP456 variant, and uncoated wells at a 1:10 serum dilution.

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[0145] While the foregoing compositions and methods have been described in some detail by way of illustration and example for purposes of clarity of understanding, these illustrations and examples should not be construed as limiting the scope of the present disclosure. The disclosures of all patent and scientific literature cited herein are expressly incorporated by reference in their entirety. SEQUENCE LISTING <110> F. HOFFMANN-LA ROCHE AG GENENTECH, INC. <120> HINGE MODIFIED ANTIBODY FRAGMENTS AND METHODS OF MAKING <130> 00B206.0271 <140> <141> <150> 62 / 346,905 <151> 2016-06-07 <150> 62 / 248,792 <151> 2015-10-30 <160> 59 <170> PatentIn version 3.5 <210> 1 <211> 108 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 1 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 100 105 <210> 2 <211> 108 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 2 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Leu 100 105 <210> 3 <211> 107 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 3 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His 100 105 <210> 4 <211> 106 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 4 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 100 105 <210> 5 <211> 105 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 5 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys 100 105 <210> 6 <211> 104 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 6 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp 100 <210> 7 <211> 101 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 7 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Thr Val Glu Arg Lys 100 <210> 8 <211> 105 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 8 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro 100 105 <210> 9 <211> 103 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 9 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly 100 <210> 10 <211> 104 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 10 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro 100 <210> 11 <211> 105 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 11 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro 100 105 <210> 12 <211> 102 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 12 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr 100 <210> 13 <211> 101 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 13 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys 100 <210> 14 <211> 6 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 14 Cys Asp Lys Thr His Thr 1 5 <210> 15 <211> 6 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 15 Cys Asp Lys Thr His Leu 1 5 <210> 16 <211> 5 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 16 Cys Asp Lys Thr His 1 5 <210> 17 <211> 4 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 17 Cys Asp Lys Thr 1 <210> 18 <211> 5 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 18 Lys Tyr Gly Pro Pro 1 5 <210> 19 <211> 4 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 19 Lys Tyr Gly Pro 1 <210> 20 <211> 5 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 20 Asp Lys Thr His Leu 1 5 <210> 21 <211> 5 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 21 Asp Lys Thr His Thr 1 5 <210> 22 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 22 Glu Pro Lys Ser Cys Asp Lys Thr His Thr 1 5 10 <210> 23 <211> 4 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 23 Cys Pro Pro Cys 1 <210> 24 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 24 Pro Ala Pro Glu Leu Leu Gly Gly Pro 1 5 <210> 25 <211> 6 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <220> <221> MOD_RES <222> (6)..(6) <223> Any amino acid except Thr <400> 25 Cys Asp Lys Thr His Xaa 1 5 <210> 26 <211> 4 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 26 Val Glu Arg Lys 1 <210> 27 <211> 4 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 27 Asp Lys Thr His 1 <210> 28 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 28 Gly His His His His His His 1 5 <210> 29 <211> 5 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 29 His His His His His 1 5 <210> 30 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 30 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 <210> 31 <211> 15 <212> PRT <213> Homo sapiens <400> 31 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 1 5 10 15 <210> 32 <211> 14 <212> PRT <213> Homo sapiens <400> 32 Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val 1 5 10 <210> 33 <211> 15 <212> PRT <213> Homo sapiens <400> 33 Cys Pro Arg Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 1 5 10 15 <210> 34 <211> 15 <212> PRT <213> Homo sapiens <400> 34 Cys Pro Ser Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val 1 5 10 15 <210> 35 <211> 23 <212> PRT <213> Homo sapiens <400> 35 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 1 5 10 15 Pro Ala Pro Glu Leu Leu Gly 20 <210> 36 <211> 14 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 36 Cys Pro Pro Cys Pro Ala Pro Glu Leu Gly Gly Pro Ser Val 1 5 10 <210> 37 <211> 14 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 37 Cys Pro Pro Cys Pro Ala Pro Leu Leu Gly Gly Pro Ser Val 1 5 10 <210> 38 <211> 14 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 38 Cys Pro Pro Cys Pro Ala Glu Leu Leu Gly Gly Pro Ser Val 1 5 10 <210> 39 <211> 13 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 39 Cys Pro Pro Cys Pro Ala Pro Leu Gly Gly Pro Ser Val 1 5 10 <210> 40 <211> 13 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 40 Cys Pro Pro Cys Pro Ala Leu Leu Gly Gly Pro Ser Val 1 5 10 <210> 41 <211> 12 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 41 Cys Pro Pro Cys Pro Leu Leu Gly Gly Pro Ser Val 1 5 10 <210> 42 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 42 Cys Pro Pro Cys Leu Leu Gly Gly Pro Ser Val 1 5 10 <210> 43 <211> 23 <212> PRT <213> Homo sapiens <400> 43 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 1 5 10 15 Pro Glu Leu Leu Gly Gly Pro 20 <210> 44 <211> 26 <212> PRT <213> Macaca fascicularis <400> 44 Glu Ile Lys Thr Cys Gly Gly Gly Ser Lys Pro Pro Thr Cys Pro Pro 1 5 10 15 Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 20 25 <210> 45 <211> 26 <212> PRT <213> Macaca mulatta <400> 45 Glu Ile Lys Thr Cys Gly Gly Gly Ser Lys Pro Pro Thr Cys Pro Pro 1 5 10 15 Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 20 25 <210> 46 <211> 19 <212> PRT <213> Homo sapiens <400> 46 Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Pro Pro Pro Val 1 5 10 15 Ala Gly Pro <210> 47 <211> 19 <212> PRT <213> Macaca fascicularis <400> 47 Gly Leu Pro Cys Arg Ser Thr Cys Pro Pro Cys Pro Ala Glu Leu Leu 1 5 10 15 Gly Gly Pro <210> 48 <211> 19 <212> PRT <213> Macaca mulatta <400> 48 Gly Leu Pro Cys Arg Ser Thr Cys Pro Pro Cys Pro Ala Glu Leu Leu 1 5 10 15 Gly Gly Pro <210> 49 <211> 67 <212> PRT <213> Homo sapiens <400> 49 Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr Cys Pro Glu Pro 1 5 10 15 Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro Glu Pro Lys 20 25 30 Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro Glu Pro Lys Ser 35 40 45 Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro Ala Pro Glu Leu Leu 50 55 60 Gly Gly Pro 65 <210> 50 <211> 25 <212> PRT <213> Macaca fascicularis <400> 50 Glu Phe Thr Arg Pro Cys Asp Asp Thr Thr Pro Pro Cys Pro Pro Cys 1 5 10 15 Pro Ala Pro Glu Leu Leu Gly Gly Pro 20 25 <210> 51 <211> 25 <212> PRT <213> Macaca mulatta <400> 51 Glu Phe Thr Pro Pro Cys Gly Asp Thr Thr Pro Pro Cys Pro Pro Cys 1 5 10 15 Pro Ala Pro Glu Leu Leu Gly Gly Pro 20 25 <210> 52 <211> 20 <212> PRT <213> Homo sapiens <400> 52 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe 1 5 10 15 Leu Gly Gly Pro 20 <210> 53 <211> 18 <212> PRT <213> Macaca fascicularis <400> 53 Glu Phe Thr Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro <210> 54 <211> 18 <212> PRT <213> Macaca mulatta <400> 54 Glu Phe Thr Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro <210> 55 <211> 70 <212> PRT <213> Homo sapiens <400> 55 Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr Cys Pro Arg Cys 1 5 10 15 Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro 20 25 30 Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro Glu 35 40 45 Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro Ala Pro 50 55 60 Glu Leu Leu Gly Gly Pro 65 70 <210> 56 <211> 16 <212> PRT <213> Homo sapiens <400> 56 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 1 5 10 15 <210> 57 <211> 13 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 57 Thr Cys Pro Pro Cys Pro Leu Leu Gly Gly Pro Ser Val 1 5 10 <210> 58 <211> 12 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 58 Thr Cys Pro Pro Cys Leu Leu Gly Gly Pro Ser Val 1 5 10 <210> 59 <211> 14 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 59 Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val 1 5 10

Claims

1. A composition comprising an isolated antibody fragment, said antibody fragment having reduced or no reactivity with pre-existing anti-hinge antibodies.

2. The antibody fragment may be Fab, Fab', or F(ab'). ２ 2. The composition of claim 1, wherein:

3. The composition of claim 2 , wherein the antibody fragment is a Fab.

4. The Fab is a Fab having residues D ２２１ The composition of claim 3 , wherein the amino acid is substituted or unsubstituted.

5. The Fab is a Fab having residue K ２２２ The composition of claim 3 , wherein the amino acid is substituted or unsubstituted.

6. 4. The composition of claim 3, wherein the Fab comprises a T225X mutation, where X is any amino acid except T.

7. 4. The composition of claim 3, wherein the Fab terminates with an amino acid comprising an amino acid sequence selected from the group consisting of CDKTHT (SEQ ID NO: 14), CDKTHL (SEQ ID NO: 15), CDKTH (SEQ ID NO: 16), CDKT (SEQ ID NO: 17), CDK, and CD.

8. 4. The composition of claim 3, wherein the Fab terminates with an amino acid comprising an amino acid sequence selected from the group consisting of KYGPP (SEQ ID NO: 18), KYGP (SEQ ID NO: 19), KYG, KY, and K.

9. 4. The composition of claim 3, wherein the Fab comprises a heavy chain constant region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and conservative modifications thereof.

10. The antibody fragment is F(ab') ２ The composition of claim 2, wherein

11. The F(ab') ２ The composition of claim 7, wherein the C-terminal amino acid sequence of the nucleotide ...

12. The F(ab') ２ The composition of claim 8, wherein the amino acid sequence comprises a deletion at EU position 231.

13. The F(ab') ２ The composition of claim 8, wherein the amino acid sequence of the present invention is a sequence similar to that of the amino acid sequence of the present invention.

14. The F(ab') ２ The composition of claim 8, wherein the gene encoding the gene for the present invention comprises a deletion in EU231-233.

15. The F(ab') ２ The composition of claim 8, wherein the gene encoding the gene for the present invention comprises a deletion in EU231-234.

16. The F(ab') ２ The composition of claim 8, wherein the gene encoding the gene for the present invention comprises a deletion in EU230-234.

17. The composition of claim 1, wherein the isolated antibody fragment exhibits reduced binding to FcγRIIIa, C1q, or a combination thereof.

18. A composition comprising an isolated nucleic acid encoding the antibody fragment of claim 1.

19. 20. A host cell comprising the composition of claim 18.

20. 20. A method for producing an antibody fragment, comprising culturing the host cell of claim 19 so that the antibody fragment is produced.

21. A pharmaceutical formulation comprising the composition of any one of claims 1 to 17 and a pharmaceutically acceptable carrier.

22. A composition according to any one of claims 1 to 17 for use as a medicament.

23. A composition according to any one of claims 1 to 17 for use in the treatment of a disease.

24. A composition according to any one of claims 1 to 17 for use in inhibiting molecular pathways and / or mechanisms.

25. Use of a composition according to any one of claims 1 to 17 in the manufacture of a medicament for the treatment of a disease.

26. Use of a composition according to any one of claims 1 to 17 in the manufacture of a medicament for the inhibition of molecular pathways and / or mechanisms.

27. Use of a composition according to any one of claims 1 to 17 in the manufacture of a medicament for the activation of molecular pathways and / or mechanisms.

28. A method of treating an individual having a disease, the method comprising administering to said individual an effective amount of a composition according to any one of claims 1 to 17.

29. 20. A method of inhibiting a molecular pathway and / or mechanism in an individual, the method comprising administering to said individual a composition of any one of claims 1 to 17 in an amount effective to inhibit the molecular pathway and / or mechanism.

30. 20. A method of activating a molecular pathway and / or mechanism in an individual, the method comprising administering to said individual a composition according to any one of claims 1 to 17 in an amount effective to activate the molecular pathway and / or mechanism.

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