Bispecific HIV-1 neutralizing antibodies
阅读说明：本技术 双特异性hiv-1中和抗体 (Bispecific HIV-1 neutralizing antibodies ) 是由 大卫·D·何 黄耀星 于健 于 2018-12-20 设计创作，主要内容包括：在各个实施方案中,本发明总体上涉及使用双特异性抗体来预防和治疗HIV。(In various embodiments, the present invention relates generally to the use of bispecific antibodies for the prevention and treatment of HIV.)
1. A bispecific antibody in CrossMab format capable of neutralizing HIV, wherein the antibody comprises light and heavy chain portions of a first antibody 10E8 or a variant thereof that binds to HIV envelope protein; and light and heavy chain portions of a second antibody ibalizumab or a variant thereof, which binds to a cell membrane receptor protein or a cell membrane co-receptor protein, wherein
The light chain portion of the first antibody 10E8 comprises a heavy chain variable region identical to SEQ ID NO:33, wherein SEQ ID NO:33 incorporates 1-4 mutations and the heavy chain portion of the first antibody 10E8 comprises a heavy chain variable region identical to SEQ ID NO:34, wherein SEQ ID NO:34 incorporates 4-12 mutations; and the light chain portion of the second antibody ibalizumab comprises an amino acid sequence identical to SEQ ID NO:1 and the heavy chain portion of the second antibody ibalizumab comprises an amino acid sequence at least 97% identical to SEQ ID NO:2 has an amino acid sequence of at least 97%; and wherein relative to SEQ ID NO: 1.2, 33 and 34 are within the variable region.
2. The bispecific antibody of claim 1, wherein SEQ ID NO:33 is an amino acid position selected from L15, P40, I45 and/or P112.
3. The bispecific antibody of claim 1, wherein SEQ ID NO:33 is an amino acid position selected from P40 and I45.
4. The bispecific antibody of claim 1, wherein SEQ ID NO:33 are P40T and I45K.
5. The bispecific antibody of claim 1, wherein SEQ ID NO:34 is an amino acid position selected from L72, I75, F77, L89, Y98, F100a, W100b, Y100e, P100F, P100g, L108 and/or L170.
6. The bispecific antibody of claim 1, wherein SEQ ID NO:34 is an amino acid position selected from L72, I75, F77 and/or L108.
7. The bispecific antibody of claim 1, wherein SEQ ID NO:34 is L72K, I75K, F77T and/or L108K.
8. The bispecific antibody of claim 1, wherein the antibody comprises a light chain portion of an ibalizumab antibody comprising the amino acid sequence of SEQ ID NO: 1.
9. The bispecific antibody of claim 1, wherein the antibody comprises a heavy chain portion of an ibalizumab antibody comprising the amino acid sequence of SEQ ID NO: 2.
10. A pharmaceutical composition comprising the bispecific antibody of claim 1 and a pharmaceutically acceptable carrier.
11. The pharmaceutical composition of claim 10, wherein the composition is formulated for oral, intranasal, pulmonary, intradermal, transdermal, subcutaneous, intramuscular, intraperitoneal, or intravenous delivery.
Technical Field
In various embodiments, the present invention relates generally to the use of bispecific antibodies for the prevention and treatment of HIV.
Sequence listing
This application contains a sequence listing that has been filed in ASCII format through EFS-Web and is hereby incorporated by reference in its entirety. The ASCII copy created on 12/21/2017 was named ADR-001CP _ st25.txt and was 145,851 bytes in size.
Background
Passive immunization with antibodies (abs) is a well-established method for the prevention and treatment of infectious diseases. Such methods may involve preparing human immunoglobulins from donors recovered from infectious diseases and using such preparations containing abs specific for the infectious organism to protect the recipient against the same disease. Alternatively, therapeutic antibodies can be made by immunizing mice with antigen, and then engineering/humanizing the mouse abs into human form. Monoclonal antibodies (mabs) are homogeneous in physical characteristics and immunochemical reactivity and thus offer the possibility of absolutely specific activity.
This specificity may ultimately be a limitation of some targets, and therefore practitioners have developed "bispecific" mabs that are composed of fragments of two different mabs and bind to two different antigen types. This facilitates, for example, binding to antigens that are only weakly expressed. Some bispecific mabs can stimulate strong immune responses, thereby limiting their clinical applications. A recent approach to improve this effect is the "CrossMab" approach, a bispecific antibody format that employs a more natural antibody-like structure.
The prospect of generating highly potent bispecific or bivalent antibodies against pathogens such as HIV for clinical applications involves a number of uncertainties. For example, low spike density and spike structure on HIV may prevent bivalent binding of antibodies to HIV, and the geometry and spatial relationship of cell surface anchoring is not well characterized. It is also unknown whether sufficient epitope accessibility exists on the HIV envelope. CrossMab bispecific antibodies anchored to host cell membranes offer the following possibilities: improved local antibody concentration, targeting of sequential and/or interdependent entry steps, and compensation for monovalent binding.
Further, large-scale commercial production of antibodies remains challenging. For example, the production of therapeutic antibodies typically requires the use of very large scale cell cultures under good production practices followed by extensive purification steps, resulting in extremely high production costs. Other limitations such as poor insolubility, protein aggregation and protein instability may also make antibody production less desirable.
Thus, there remains a need for therapeutically effective HIV antibodies that can be readily produced on a commercial scale.
Disclosure of Invention
In one aspect, the invention relates to a bispecific antibody for neutralizing HIV. The bispecific antibody comprises portions of a first antibody and a second antibody, wherein the first antibody binds to an HIV envelope protein. In certain embodiments, the first antibody is selected from the group consisting of PGT145, PG9, PGT128, PGT121, 10-1074, 3BNC117, VRC01, PGT151, 4E10, 10E8, and variants thereof. In certain embodiments, the bispecific antibody comprises a portion of a second antibody, wherein the second antibody binds to a cell membrane protein. For example, the second antibody binds to a cell receptor protein or a cell membrane co-receptor protein. In one embodiment, the second antibody is selected from the group consisting of a CD4 antibody, a CCR5 antibody, and a CXCR4 antibody, such as Pro140, ibalizumab (ibalizumab), 515H7, or a variant thereof. In various embodiments, the bispecific antibody has a CrossMab format.
In another aspect, the invention provides a bispecific antibody comprising portions of a first antibody and a second antibody, wherein the first antibody binds to HIV envelope proteins and the second antibody binds to cell membrane proteins. In various embodiments, the bispecific antibody has a CrossMab format.
In various embodiments, pharmaceutical compositions are also provided comprising the bispecific antibodies disclosed herein. The pharmaceutical composition may be formulated for oral, intranasal, pulmonary, intradermal, transdermal, subcutaneous, intramuscular, intraperitoneal or intravenous delivery.
In another aspect, a method for neutralizing HIV is provided. The method comprises the following steps: the antigen binding site is contacted with a bispecific antibody that binds to an HIV envelope protein and the other antigen binding site is contacted with a bispecific antibody that binds to a cell membrane protein.
In another aspect, methods for treating a patient infected with HIV are also provided. The method comprises administering to the patient any of the bispecific antibodies or pharmaceutical compositions as disclosed herein. In one embodiment, the patient is a human.
Drawings
In the drawings, like reference numerals generally refer to the same parts throughout the different views. Furthermore, the drawings are not necessarily to scale, emphasis instead generally being placed upon illustrating the principles of the invention. In the following description, various embodiments of the present invention are described with reference to the following drawings, in which:
figure 1 is a graph showing CrossMab antibodies derived from two IgG monoclonal antibodies.
FIG. 2A is a graph showing iMab antibody targeting CD4 (short for monoclonal antibody ibalizumab) and Pro140
FIG. 2B is a diagram showing mAbs targeting
Fig. 3 is a graph comparing the percent maximal inhibition of anti-cell to cell HIV transmission (MPI) using iMab in combination with the 10E8 antibody with CrossMab bispecific 10E8/iMab antibody. All iMab-based bispecific antibodies were constructed using the MV1 variant, unless otherwise indicated.
FIGS. 4A-J are a series of graphs comparing inhibition against various strains of X4 and amphotropic HIV using different concentrations of 10E8, Pro140, or 10E8/P140 antibody. P140 is short for Pro 140.
Fig. 5A-G are a series of graphs comparing inhibition against various HIV strains using different concentrations of 10E8, Pro140, 10E8/P140, or individual 10E8 in combination with Pro140 monoclonal antibodies.
FIGS. 6A-D are a series of graphs comparing inhibition against various HIV strains using different concentrations of 10E8, X19, 10E8/X19, or 10E8/P140 antibody.
FIGS. 7A-H are a series of graphs comparing inhibition against various HIV strains using different concentrations of the 10E8, Pro140, 10E8/P140, and 10E8/α Her2 antibodies.
FIG. 8A is a graph comparing the binding of the CrossMab bispecific antibody 10E8/iMab and Δ 10E8/iMab to the HIV-1 glycoprotein MPER.
Fig. 8B-E are a series of graphs comparing the percent inhibition of 10E8 (light gray line) and Δ 10E8 (dark gray line) against iMab resistant R5 virus (fig. 8B) and X4 virus (fig. 8C) and 10E8/iMab (light gray line) and Δ 10E8/iMab (dark gray line) against iMab resistant R5 virus (fig. 8D) and X4 virus (fig. 8E).
FIGS. 9A-G are a series of graphs comparing inhibition against various HIV strains using different concentrations of 10E8, Δ 10E8, 4E10, 10E8/P140, Δ 10E8/P140, and 4E10/P140 antibodies.
FIG. 10 is a graph comparing the antiviral coverage of CrossMab antibodies 10E8/Pro140 and 10E8/iMab, their parental monoclonal antibodies 10E8, Pro140 and iMab, and various other HIV envelope-targeting monoclonal antibodies against a large panel of HIV envelope pseudoviruses.
FIGS. 11A-E are a series of graphs comparing the Maximum Percent Inhibition (MPI) of monoclonal antibody iMab (grey bars in all figures) and the
FIGS. 12A-E are a series of graphs comparing the Maximum Percent Inhibition (MPI) and IC80 antibody concentrations for a bulk HIV envelope pseudotyped virus of
FIGS. 13A-E are a series of graphs comparing IC80 antibody concentrations for the crossMab bispecific antibody based on iMab and Pro140 and its parent antibodies PGT145/iMab and PGT145/Pro140 (FIG. 13A), 3BNC117/iMab and 3BNC117/Pro140 (FIG. 13B), PGT128/iMab and PGT128/Pro140 (FIG. 13C), PGT151/iMab and PGT151/Pro140 (FIG. 13D), and 10E8/iMab and 10E8/Pro140 (FIG. 13E).
FIGS. 14A-E are a series of graphs comparing IC50 antibody concentrations for the crossMab bispecific antibodies based on iMab and Pro140 and their parent antibodies PGT145/iMab and PGT145/Pro140 (FIG. 14A), 3BNC117/iMab and 3BNC117/Pro140 (FIG. 14B), PGT128/iMab and PGT128/Pro140 (FIG. 14C), PGT151/iMab and PGT151/Pro140 (FIG. 14D), and 10E8/iMab and 10E8/Pro140 (FIG. 14E).
FIGS. 15A-E are graphs showing the IC80 antibody concentrations for the crossMab bispecific antibody based on iMab and its parent antibodies 10E8/iMab (FIG. 15A), 3BNC117/iMab (FIG. 15B), PGT145/iMab (FIG. 15C), PGT128/iMab (FIG. 15D), and PGT151/iMab (FIG. 15E) against cell-to-cell transmission of HIV.
Fig. 16 is a graph demonstrating the Maximum Percent Inhibition (MPI) of the cell-to-cell transmission of CrossMab bispecific antibody and its parent antibody against HIV.
Figure 17A is a graph comparing inhibition against HIV strains of different concentrations of 10E8, Pro140, 10E8/P140CrossMab bispecific antibody and combinations of individual 10E8 and Pro140 monoclonal antibodies.
Fig. 17B is a graph comparing the inhibition of different concentrations of iMab, 10E8, 10E8/iMab CrossMab bispecific antibody and individual combinations of 10E8 and iMab monoclonal antibodies against HIV strains.
FIGS. 18A-D are a series of graphs comparing inhibition of different concentrations of the 10E8, Pro140, 10E8/P140, and 10E8/515H7 antibodies against various HIV R5 strains.
Fig. 18E-H are a series of graphs comparing inhibition of various concentrations of the 10E8, 515H7, and 10E8/515H7 antibodies against various HIV X4 strains.
FIGS. 19A-B are a series of graphs comparing the inhibition of different concentrations of the 10E8/Pro140, 10E8/iMab, 10E8/515H7, and 10E8/X19 antibodies against various HIV strains.
Figure 19C indicates the density of CD4, CCR5, and CXCR4 receptors present on TZM-bl cells.
FIG. 20 compares the binding of the CrossMab bispecific antibodies 10E8/Pro140, Δ 10E8/Pro140 and 4E10/Pro140 to the HIV-1 glycoprotein MPER.
FIGS. 21A-G are a series of graphs comparing the inhibition of different concentrations of the 4E10, Pro140 and 4E10/P140 and 10E8/P140 antibodies against various HIV strains.
FIG. 22A is a size exclusion chromatographic analysis of the CrossMab antibodies 10E8/iMab, 10E8/P140, and
FIG. 22B is a size exclusion chromatographic analysis of monoclonal antibodies iMab, 10E8 and
FIG. 23 is a size exclusion chromatographic analysis of monoclonal antibody 10E8 and a chimeric antibody consisting of a 10E8 heavy chain paired with a 4E10 light chain.
Fig. 24A-C are a series of size exclusion chromatograms of monoclonal antibodies 10E8 and 4E10 and chimeric antibodies consisting of a 10E8 heavy chain paired with a 4E10 light chain (fig. 24A), monoclonal antibodies 10E8 and 10E8 mutants with a potential stabilizing mutation engineered in the 10E8 light chain (fig. 24B), and monoclonal antibodies 10E8 and 10E8 mutants genetically grafted with a kappa light chain of a non-10E 8 antibody (fig. 24C).
FIG. 25 is a size exclusion chromatogram of monoclonal antibody 4E10 and a 4E10 mutant gene grafted with the light region of 10E8 (including CDR1 region, CDR2 region, CDR3 region or combined CDR1, CDR2 and CDR3 regions).
Fig. 26A is a size exclusion chromatogram of a 10E8 chimeric antibody. CDR123 is a chimeric antibody in which the 10E8 heavy chain is paired with the 10E8 light chain gene-grafted with the 10E8 antibody germ line CDR region sequences. FW123 is a chimeric antibody in which the 10E8 heavy chain is paired with the 10E8 light chain gene-grafted with the 10E8 antibody germ line framework region sequence.
Fig. 26B is a table indicating expression of CDR123 and FW123 antibodies, HIV MPER binding capacity, size exclusion chromatography profiles, and HIV neutralization profiles.
Figure 27 is a size exclusion chromatogram of monoclonal antibody 10E8, its somatic variant H6L10, and a CrossMab bispecific antibody consisting of H6L10 paired with
FIG. 28 is a graph depicting the pharmacokinetic profile of 10E8, H6L10/Pro140 and their parent antibodies in a mouse model.
FIG. 29 shows a comparison of 10E8v1.0Graph of the potency of the/iMab or P140CrossMab antibodies versus the 10E8/iMab or P140 antibodies.
Fig. 30 is a graph depicting the pharmacokinetics of 10E8 and CrossMab antibodies derived from several 10E8 variants and iMab or P140 in a mouse model.
FIGS. 31A-B depict 10E8v1.1/P140 and 10E8v2.0A series of graphs of HIV viral coverage of/iMab antibodies.
FIGS. 31C-D depict 10E8v1.1/P140 and 10E8v2.0A series of figures of size exclusion chromatographic stability profiles of/iMab antibodies.
FIGS. 32A-B depict 10E8 stored in PBS at 4 ℃v1.1/P140 and 10E8v2.0A series of graphs of size exclusion stability profiles of/iMab antibodies.
FIG. 33 depicts 10E8v2.0Native mass spectrometry analysis of the/iMab (N297A) antibody.
FIGS. 34A-C are comparative 10E8v1.1/P140 and 10E8v2.0A series of plots of the activity of/iMab on HIV arm group C and the IC50 and IC80 activities of these antibodies.
FIGS. 35 and 36 compare 10E8v1.1/P140、10E8v2.0Graphs of the efficacy of/iMab and various monoclonal antibodies against HIV.
FIGS. 37A-C show that a select number of 10E8V2.0/iMab (also referred to as 10E8.2/iMab) variants retained functional antiviral activity and increased solubility. FIG. 37A shows that some 10E8.2/iMab variants retain functional activity in an in vitro HIV-1 neutralization assay. FIG. 37B shows that 10E8.2/iMab and some 10E8.2/iMab variants have similar in vivo pharmacokinetic profiles. FIG. 37C shows precipitation profiles of 10E8.2/iMab and some 10E8.2/iMab variants under heat stress-induced conditions.
FIGS. 38A-B show the results of size exclusion chromatography used to identify 10E8.2/iMab variants with minimal aggregation after heat stress induced conditions.
FIGS. 39A-B show the solubilities of the 10E8.2/iMab and 10E8.4/iMab variants at 4 ℃ after ultracentrifugation.
FIG. 40 shows the turbidity as a function of time for different concentrations of the 10E8.2/iMab and 10E8.4/iMab variants.
FIG. 41 shows the thermal stability of the 10E8.2/iMab and 10E8.4/iMab variants as assessed by differential scanning calorimetry.
FIG. 42 shows the turbidity of the 10E8.2/iMab and 10E8.4/iMab variants after forced degradation at 50 ℃ for six days. For each set of histograms, bars from left to right represent 10E8.2/iMab (before centrifugation), 10E8.4/iMab (before centrifugation), 10E8.2/iMab (after centrifugation), and 10E8.4/iMab (after centrifugation).
FIG. 43 shows anti-HIV coverage of 10E8.2/iMab and 10E8.4/iMab variants.
FIG. 44 is a graph showing the in vivo antiviral activity of 10E8.2/iMab and 10E8.4/iMab variants in a humanized mouse model of HIV-1 infection.
FIG. 45A shows a sequence alignment of the light chain of 10E8.2/iMab (SEQ ID NO:33) with the light chain of the 10E8.4/iMab variant (SEQ ID NO: 44).
FIG. 45B shows a sequence alignment of the heavy chain of 10E8.2/iMab (SEQ ID NO:34) with the heavy chain of the 10E8.4/iMab variant (SEQ ID NO: 42). Underlined sequences represent CDR1, CDR2, and
Fig. 46 is a graph showing exemplary variants of the 10E8 antibody that are stable while retaining anti-HIV activity.
Detailed Description
Embodiments of the present invention provide for the inhibition of HIV. In various implementations, bispecific antibodies are formed, each comprising a heavy chain component and a light chain component from two different parent antibodies. In various embodiments, one parent antibody specifically binds HIV, e.g., the HIV envelope protein Env. In various embodiments, the other parent antibody specifically binds to a cell membrane protein, such as CD4 and
In various embodiments, bispecific antibodies of the invention (e.g., HIV CrossMab antibodies) have the native structure of an IgG molecule, but are bispecific. In one bispecific antibody, the heavy and light chains from each of the two parent antibodies are combined, thereby providing an antibody in which the antigen binding sites of fragment antigen binding 1(Fab1) and Fab2 have different binding specificities. In certain embodiments, the bispecific antibody is a CrossMab format antibody as shown in figure 1. In CrossMab format, one heavy chain comprises a "knob" structure and the other heavy chain comprises a corresponding "hole" structure, and the positions of the constant domains (i.e., CL and CH1) from one parent antibody are swapped, together ensuring proper pairing of the heavy and light chains during assembly.
Various mabs have been shown to block HIV infection by targeting and binding to the HIV envelope protein Env (fig. 2B and fig. 10). These mabs include, for example, PGT145, PG9, PGT128, PGT121, 10-1074, 3BNC117, VRC01, PGT151, 4E10, and 10E8. FIG. 2B (adapted from www.scripps.edu/news/press/2014/20140424HIV. html) shows how mAbPGT145 targets the V1/V2 epitope on the envelope gp120 of the HIV virus; how mAb PGT128 targets glycans on the V3 stem region of
In addition, monoclonal antibodies Pro140 ("P140"), ibalizumab ("iMab"), and 515H7 have been shown to block HIV infection by targeting and binding to CCR5, CD4, and CXCR4 human cell membrane proteins, respectively (fig. 2A). In particular, fig. 2A shows how iMab targets CD4, the major receptor for HIV-1 entry expressed on human T cells; and how Pro140 targets CCR5, a co-receptor for HIV-1 entry through CCR5 tropic HIV-1.
While the discussion that follows focuses on the use of bispecific antibodies against Env and cell membrane proteins CD4 and CCR5, it will be understood that this is for ease of presentation only and that any suitable antibody against any HIV epitope and any suitable antibody against any suitable cell membrane protein may be used and are within the scope of the present invention.
Thus, in various embodiments, the present invention provides bispecific antibodies that target and bind to HIV Env proteins as well as cell membrane proteins CCR5, CD4, and/or
Amino acid sequences defining the heavy and light chains of the PGT145 antibody may be found, for example, at www.ncbi.nlm.nih.gov/protein/3U1S _ H and http:// www.ncbi.nlm.nih.gov/protein/3U1S _ L, respectively, the entire contents of which are incorporated herein by reference.
Amino acid sequences defining the heavy and light chains of the PG9 antibody can be found, for example, at www.ncbi.nlm.nih.gov/protein/3U4E _ H and www.ncbi.nlm.nih.gov/protein/3MUH _ L, respectively, which are incorporated herein by reference in their entirety.
Amino acid sequences defining the heavy and light chains of the PGT128 antibody can be found, for example, at www.ncbi.nlm.nih.gov/protein/3TYG _ H and www.ncbi.nlm.nih.gov/protein/3TYG _ L, respectively, the entire contents of which are incorporated herein by reference.
Amino acid sequences defining the heavy and light chains of the PGT121 antibody may be found, for example, at www.ncbi.nlm.nih.gov/protein/4FQC _ H and www.ncbi.nlm.nih.gov/protein/4FQC _ L, respectively, the entire contents of which are incorporated herein by reference.
Amino acid sequences defining the heavy and light chains of the 10-1074 antibody can be found, for example, in Mouquet h. et al, (2012) PNAS,109(47) E3268-77 (including supplementary information), the entire contents of which are incorporated herein by reference.
Definitions the amino acid sequences of the heavy and light chains of the 3BNC117 antibody can be found, for example, at www.ncbi.nlm.nih.gov/protein/4LSV _ H and www.ncbi.nlm.nih.gov/protein/4LSV _ L, respectively, which are incorporated herein by reference in their entirety.
Amino acid sequences defining the heavy and light chains of the VRC01 antibody can be found, for example, at www.ncbi.nlm.nih.gov/protein/4LST _ H and www.ncbi.nlm.nih.gov/protein/4LST _ L, respectively, which are incorporated herein by reference in their entirety.
Amino acid sequences defining the heavy and light chains of the PGT151 antibody may be found, for example, at www.ncbi.nlm.nih.gov/protein/4NUG _ H and www.ncbi.nlm.nih.gov/protein/4NUG _ L, respectively, the entire contents of which are incorporated herein by reference.
Definitions the amino acid sequences of the heavy and light chains of the 4E10 antibody may be found, for example, at www.ncbi.nlm.nih.gov/protein/4LLV _ H and www.ncbi.nlm.nih.gov/protein/4LLV _ L, respectively, which are incorporated herein by reference in their entirety.
Amino acid sequences defining the heavy and light chains of the 10E8 antibody can be found, for example, at www.ncbi.nlm.nih.gov/protein/4G6F _ B and www.ncbi.nlm.nih.gov/protein/4G6F _ D, respectively, which are incorporated herein by reference in their entirety.
In certain embodiments, bispecific antibodies include sequences (e.g., heavy chain and light chain sequences) derived from, but not limited to, P140, iMab (or MV1 variant), and/or 515H7 antibodies and variants thereof. The heavy and light chain sequences of the Pro140, iMab (or MV1 variants thereof), and 515H7 antibodies are further described, for example, in Olson, w.c. et al, (1999) jvirol, 73(5) 4145-55; trkola, a. et al, (2001) J virol, 75(2) 579-88; U.S. patent nos. 7,122,185; burkly l.c. et al, (1992) J immunol.,149(5) 1779-87; moore j.p. et al, (1992) J virol, 66(8) 4784-93; reimann K.A. et al, (1997) AIDS Res Hum Retroviruses, 13(11): 933-43; international patent publication No. WO2014100139 and european patent publication No. EP2246364, the entire contents of which are incorporated herein by reference.
As used herein, an antibody "variant" refers to an antibody having an amino acid sequence that is different from the amino acid sequence of the parent antibody from which it is derived. In various embodiments, the variant has one or more amino acid changes relative to the parent antibody.
In various embodiments, the bispecific antibodies of the invention comprise a heavy chain sequence and a light chain sequence from the PGT145, PG9, PGT128, PGT121, 10-1074, 3BNC117, VRC01, PGT151, 4E10 or 10E8 antibody or variants thereof and a heavy chain sequence and a light chain sequence from the P140, iMab (or MV1 variant) or 515H7 antibody or variants thereof.
In an exemplary embodiment, a series of HIV CrossMab antibodies have been constructed, including but not limited to, for example, 145/MV1, 117/MV1, 128/MV1, 10E8/MV1, 145/P140, 128/P140, 117/P140, 10E8/P140, 10E8/α -Her2, 10E8/X19, and
Amino acid sequences defining the heavy and light chains of various exemplary HIV CrossMab antibodies are shown below.
145/MV1 antibody:
amino acid sequence defining MV 1-derived light chain of 145/MV1 antibody-MV 1-VLCH1(SEQ ID NO: 1):
amino acid sequence defining the MV 1-derived heavy chain of the 145/MV1 antibody-MV 1-HC-mortar-crossing (Cross) (SEQ ID NO: 2):
amino acid sequence defining the PGT 145-derived light chain of the 145/MV1 antibody-PGT 145-LC (SEQ ID NO: 3):
amino acid sequence defining the PGT 145-derived heavy chain of the 145/MV1 antibody-PGT 145-HC-pestle (SEQ ID NO: 4):
117/MV1 antibody:
amino acid sequence defining the MV 1-derived light chain of the 117/MV1 antibody-MV 1-VLCH1(SEQ ID NO: 1):
amino acid sequence defining the MV 1-derived heavy chain of the 117/MV1 antibody-MV 1-HC-mortar-crossing (Cross) (SEQ ID NO: 2):
amino acid sequence defining the 3BNC 117-derived light chain of the 117/MV1 antibody-3 BNC117-LC (SEQ ID NO: 5):
amino acid sequence defining the 3BNC 117-derived heavy chain of the 117/MV1 antibody-3 BNC 117-HC-pestle (SEQ ID NO: 6):
128/MV1 antibody:
amino acid sequence defining the MV 1-derived light chain of the 128/MV1 antibody-MV 1-VLCH1(SEQ ID NO: 1):
amino acid sequence defining the MV 1-derived heavy chain of the 128/MV1 antibody-MV 1-HC-mortar-crossing (Cross) (SEQ ID NO: 2):
amino acid sequence defining the PGT 128-derived light chain of the 128/MV1 antibody-PGT 128-LC (SEQ ID NO: 7):
amino acid sequence defining the PGT 128-derived heavy chain of the 128/MV1 antibody-PGT 128-HC-pestle (SEQ ID NO: 8):
10E8/MV1 antibody:
amino acid sequence defining the MV 1-derived light chain of the 10E8/MV1 antibody-MV 1-VLCH1(SEQ ID NO: 1):
amino acid sequence defining the MV 1-derived heavy chain of the 10E8/MV1 antibody-MV 1-HC-mortar-crossing (SEQ ID NO: 2):
amino acid sequence defining 10E 8-derived light chain of 10E8/MV1 antibody-10E 8-LC (SEQ ID NO: 9):
amino acid sequence defining the 10E 8-derived heavy chain of the 10E8/MV1 antibody-10E 8-HC-pestle (SEQ ID NO: 10):
delta 10E8/MV1 antibody
Amino acid sequence MV1-VLCH1(SEQ ID NO: 1) defining the MV 1-derived light chain of the Δ 10E8/MV1 antibody:
amino acid sequence MV 1-HC-mortar-crossover (SEQ ID NO:2) defining the MV 1-derived heavy chain of the Δ 10E8/MV1 antibody:
amino acid sequence defining the Δ 10E 8-derived light chain of the Δ 10E8/MV1 antibody Δ 10E8-LC (SEQ ID NO: 21):
amino acid sequence 10E 8-HC-pestle (SEQ ID NO:22) defining the Δ 10E 8-derived heavy chain of the Δ 10E8/MV1 antibody:
151/MV1 antibody
Amino acid sequence defining MV 1-derived light chain of 151/MV1 antibody-MV 1-VLCH1(SEQ ID NO: 1):
amino acid sequence MV 1-HC-mortar-crossing (SEQ ID NO:2) defining the MV 1-derived heavy chain of the 151/MV1 antibody:
amino acid sequence PGT151-LC (SEQ ID NO:23) defining the PGT 151-derived light chain of the 151/MV1 antibody:
amino acid sequence PGT 151-HC-pestle (SEQ ID NO:24) defining the PGT 151-derived heavy chain of the 151/MV1 antibody:
145/P140 antibody:
amino acid sequence-PRO 140-VLCH1(SEQ ID NO: 11) defining the Pro 140-derived light chain of the 145/P140 antibody:
amino acid sequence defining Pro 140-derived heavy chain of 145/P140 antibody-PRO 140-HC-mortar-cross (SEQ ID NO: 12):
amino acid sequence defining PGT 145-derived light chain of 145/P140 antibody-PGT 145-LC (SEQ ID NO: 3):
amino acid sequence defining the PGT 145-derived heavy chain of the 145/P140 antibody-PGT 145-HC-pestle (SEQ ID NO: 4):
117/P140 antibody:
amino acid sequence-PRO 140-VLCH1(SEQ ID NO: 11) defining the Pro 140-derived light chain of the 117/P140 antibody:
amino acid sequence-PRO 140-HC-mortar-crossover (SEQ ID NO: 12) defining Pro 140-derived heavy chain of 117/P140 antibody:
amino acid sequence defining the 3BNC 117-derived light chain of the 117/P140 antibody-3 BNC117-LC (SEQ ID NO: 5):
amino acid sequence defining the 3BNC 117-derived heavy chain of the 117/P140 antibody-3 BNC 117-HC-pestle (SEQ ID NO: 6):
128/P140 antibody:
amino acid sequence-PRO 140-VLCH1(SEQ ID NO: 11) defining the Pro 140-derived light chain of the 128/P140 antibody:
amino acid sequence defining Pro 140-derived heavy chain of 128/P140 antibody-PRO 140-HC-mortar-cross (SEQ ID NO: 12):
amino acid sequence defining the PGT 128-derived light chain of the 128/P140 antibody-PGT 128-LC (SEQ ID NO: 7):
amino acid sequence defining the PGT 128-derived heavy chain of the 128/P140 antibody-PGT 128-HC-pestle (SEQ ID NO: 8):
10E8/P140 antibody:
amino acid sequence defining Pro 140-derived light chain of 10E8/P140 antibody-PRO 140-VLCH1(SEQ ID NO: 11):
amino acid sequence defining Pro 140-derived heavy chain of 10E8/P140 antibody-PRO 140-HC-mortar-cross (SEQ ID NO: 12):
amino acid sequence defining the 10E 8-derived light chain of the 10E8/P140 antibody-10E 8-LC (SEQ ID NO: 9):
amino acid sequence defining the 10E 8-derived heavy chain of the 10E8/P140 antibody-10E 8-HC-pestle (SEQ ID NO: 10):
delta 10E8/P140 antibody
Amino acid sequence defining the PRO 140-derived light chain of the Δ 10E8/P140 antibody-PRO 140-VLCH1(SEQ ID NO: 11):
amino acid sequence defining the PRO 140-mortar-crossover (SEQ ID NO: 12) of the PRO 140-derived heavy chain of the Δ 10E8/P140 antibody:
amino acid sequence defining the Δ 10E 8-derived light chain of the Δ 10E8/P140 antibody- Δ 10E8-LC (SEQ ID NO: 21):
amino acid sequence defining the Δ 10E 8-derived heavy chain of the Δ 10E8/P140 antibody-10E 8-HC-pestle (SEQ ID NO: 22):
151/P140 antibodies
Amino acid sequence defining PRO140-VLCH1(SEQ ID NO: 11) of the PRO 140-derived light chain of the 151/P140 antibody:
amino acid sequence-PRO 140-mortar-crossover (SEQ ID NO: 12) defining the PRO 140-derived heavy chain of the 151/P140 antibody:
amino acid sequence defining PGT 151-derived light chain of 151/P140 antibody-PGT 151-LC (SEQ ID NO: 23):
amino acid sequence defining the PGT 151-derived heavy chain of the 151/P140 antibody-PGT 151-HC-pestle (SEQ ID NO: 24):
10E8/α -Her2 antibody:
amino acid sequence defining the alpha-Her 2 derived light chain of the
amino acid sequence defining the alpha-Her 2 derived heavy chain of the
amino acid sequence defining 10E 8-derived light chain of 10E8/α -Her2 antibody-10E 8-LC (SEQ ID NO: 9):
amino acid sequence defining the 10E 8-derived heavy chain of the 10E8/α -Her2 antibody-10E 8-HC-pestle (SEQ ID NO: 10):
4E10/P140 antibody:
amino acid sequence defining Pro 140-derived light chain of 4E10/P140 antibody-PRO 140-VLCH1(SEQ ID NO: 11):
amino acid sequence defining Pro 140-derived heavy chain of 4E10/P140 antibody-PRO 140-HC-mortar-cross (SEQ ID NO: 12):
amino acid sequence defining the 4E 10-derived light chain of the 4E10/P140 antibody-4E 10-LC (SEQ ID NO: 17):
amino acid sequence defining the 4E 10-derived heavy chain of the 4E10/P140 antibody-PGT 145-HC-pestle (SEQ ID NO: 18):
10E8/X19 antibody:
amino acid sequence defining the X19-derived light chain of the 10E8/X19 antibody-X19-VLCH 1(SEQ ID NO: 19):
amino acid sequence-X19-HC-mortar-cross (SEQ ID NO:20) defining the X19-derived heavy chain of the 10E8/X19 antibody:
definition of the amino acid sequence of the 10E 8-derived light chain of the 10E8/X19 antibody-10E 8-LC (SEQ ID NO: 9):
amino acid sequence defining the 10E 8-derived heavy chain of the 10E8/X19 antibody-PGT 145-HC-pestle (SEQ ID NO: 10):
10E8/515H7 antibodies
Amino acid sequence defining the 515H 7-derived light chain of the 10E8/515H7 antibody-515H 7-VLCH 1(SEQ ID NO: 25):
amino acid sequence defining the 515H 7-derived heavy chain of the 10E8/515H7 antibody-515H 7-hole-crossing (SEQ ID NO: 26):
definition of the amino acid sequence of the 10E 8-derived light chain of the 10E8/515H7 antibody-10E 8-LC (SEQ ID NO: 9):
amino acid sequence defining the 10E 8-derived heavy chain of the 10E8/515H7 antibody-10E 8-HC-pestle (SEQ ID NO: 10):
chimeric CDR123 antibody (SEQ ID NO: 27):
chimeric FW123(SEQ ID NO: 28):
10E8V1.0/iMab antibodies
Amino acid sequence defining the MV 1-derived light chain of the 10E8v1.0/MV1 antibody-MV 1-VLCH1(SEQ ID NO: 1):
amino acid sequence defining the MV 1-derived heavy chain of the 10E8v1.0/MV1 antibody MV 1-HC-mortar-crossing (SEQ ID NO: 2):
amino acid sequence defining 10E8v1.0/iMab antibody 10E8v1.0-LC (SEQ ID NO:29) derived light chain:
definition of 10E8v1.0/iMab antibody 10E8v1.0 heavy chain derived amino acid sequence-10 E8v1.0-HC-pestle (SEQ ID NO: 30):
10E8V1.1/iMab antibodies
Amino acid sequence MV1-VLCH1(SEQ ID NO: 1) defining the MV 1-derived light chain of the 10E8v1.1/iMab antibody:
amino acid sequence MV 1-HC-mortar-cross (SEQ ID NO:2) defining the MV 1-derived heavy chain of the 10E8v1.1/iMab antibody:
definition of 10E8v1.1/iMab antibody 10E8v1.1 derived light chain of the amino acid sequence-10 E8v1.1-LC (SEQ ID NO: 31):
definition of 10E8v1.1/iMab antibody 10E8v1.1 heavy chain derived amino acid sequence-10 E8v1.1-HC-pestle (SEQ ID NO: 32):
10E8V2.0/iMab antibody (also known as 10E8.2/iMab antibody)
Amino acid sequence MV1-VLCH1(SEQ ID NO: 1) defining the MV 1-derived light chain of the 10E8v2.0/iMab antibody:
amino acid sequence MV 1-HC-mortar-crossover (SEQ ID NO:2) defining the MV 1-derived heavy chain of the 10E8v2.0/iMab antibody:
amino acid sequence defining 10E8v2.0/iMab antibody 10E8v2.0-LC (SEQ ID NO:33) derived light chain-10 E8v2.0-LC:
amino acid sequence defining the 10E8v2.0/
10E8V3.0/iMab antibodies
Amino acid sequence MV1-VLCH1(SEQ ID NO: 1) defining the MV 1-derived light chain of the 10E8v3.0/iMab antibody:
amino acid sequence MV 1-HC-mortar-crossover (SEQ ID NO:2) defining the MV 1-derived heavy chain of the 10E8v3.0/iMab antibody:
amino acid sequence defining 10E8v3.0/iMab antibody 10E8v3.0-LC (SEQ ID NO: 15) derived light chain-10 E8v3.0-LC:
amino acid sequence defining the 10E8v3.0/
10E8V1.0/P140(H6L10/PRO140) antibody
Amino acid sequence defining the PRO 140-derived light chain of the 10E8V1.0/P140 antibody-PRO 140-VLCH1(SEQ ID NO: 11):
amino acid sequence defining the PRO 140-derived heavy chain of the 10E8V1.0/P140 antibody-PRO 140-mortar-crossover (SEQ ID NO: 12):
amino acid sequence defining the L10-derived light chain of the 10E8V1.0/P140 antibody-L10-LC (SEQ ID NO: 29):
amino acid sequence defining the H6-derived heavy chain of the 10E8V1.0/P140 antibody-H6-HC-pestle (SEQ ID NO: 30):
10E8V1.1/P140 antibody
Amino acid sequence PRO140-VLCH1(SEQ ID NO: 11) defining the PRO 140-derived light chain of the 10E8v1.1/P140 antibody:
amino acid sequence PRO 140-HC-mortar-cross (SEQ ID NO: 12) defining the P140-derived heavy chain of the 10E8v1.1/P140 antibody:
definition of 10E8v1.1/P140 antibody 10E8v1.1 derived light chain of the amino acid sequence-10 E8v1.1-LC (SEQ ID NO: 31):
definition of 10E8v1.1/P140 antibody 10E8v1.1 HC-pestle (SEQ ID NO: 32):
10E8V2.0/P140 antibody
Amino acid sequence PRO140-VLCH1(SEQ ID NO: 11) defining the PRO 140-derived light chain of the 10E8v2.0/P140 antibody:
amino acid sequence PRO 140-HC-mortar-crossover defining the P140-derived heavy chain of the 10E8v2.0/P140 antibody (SEQ ID NO: 12):
amino acid sequence defining 10E8v2.0/P140 antibody 10E8v2.0-LC (SEQ ID NO:33) derived from the light chain:
amino acid sequence defining the 10E8v2.0/P140 antibody 10E8v2.0 derived heavy chain-10 E8v2.0 HC-pestle (SEQ ID NO: 34):
10E8V3.0/P140 antibody
Amino acid sequence PRO140-VLCH1(SEQ ID NO: 11) defining the PRO 140-derived light chain of the 10E8v3.0/P140 antibody:
amino acid sequence PRO 140-HC-mortar-crossover defining the P140-derived heavy chain of the 10E8v3.0/P140 antibody (SEQ ID NO: 12):
amino acid sequence defining 10E8v3.0/P140 antibody 10E8v3.0-LC (SEQ ID NO: 15) derived from the light chain:
amino acid sequence defining the 10E8v3.0/P140 antibody 10E8v3.0 derived heavy chain-10 E8v3.0 HC-pestle (SEQ ID NO: 16):
10E8.2.1/iMab
amino acid sequence MV1-VLCH1(SEQ ID NO: 1) defining the MV 1-derived light chain of the 10E8.2/iMab antibody
Definition of the amino acid sequence MV 1-HC-mortar-crossover (SEQ ID NO:2) of the MV 1-derived heavy chain of the 10E8.2/iMab antibody
Definition of 10E8.2/iMab antibody 10E8.2 derived light chain amino acid sequence-10E 8.2-LC (SEQ ID NO:33)
Amino acid sequence of the 10 E8.2.1-derived heavy chain of the 10E8.2.1/iMab antibody-10 E8.2.1-HC-pestle (SEQ ID NO:35)
10E8.2.2/iMab
Amino acid sequence MV1-VLCH1(SEQ ID NO: 1) defining the MV 1-derived light chain of the 10E8.2/iMab antibody
Definition of the amino acid sequence MV 1-HC-mortar-crossover (SEQ ID NO:2) of the MV 1-derived heavy chain of the 10E8.2/iMab antibody
Definition of 10E8.2/iMab antibody 10E8.2 derived light chain amino acid sequence-10E 8.2-LC (SEQ ID NO:33)
Amino acid sequence of the 10 E8.2.2-derived heavy chain of the 10E8.2.2/iMab antibody-10 E8.2.2-HC-pestle (SEQ ID NO:36)
10E8.2.3/iMab
Amino acid sequence MV1-VLCH1(SEQ ID NO: 1) defining the MV 1-derived light chain of the 10E8.2/iMab antibody
Definition of the amino acid sequence MV 1-HC-mortar-crossover (SEQ ID NO:2) of the MV 1-derived heavy chain of the 10E8.2/iMab antibody
Definition of 10E8.2/iMab antibody 10E8.2 derived light chain amino acid sequence-10E 8.2-LC (SEQ ID NO:33)
Amino acid sequence of the 10 E8.2.3-derived heavy chain of the 10E8.2.3/iMab antibody-10 E8.2.3-HC-pestle (SEQ ID NO:37)
10E8.2.4/iMab
Amino acid sequence MV1-VLCH1-LM52(SEQ ID NO:38) defining the MV 1-derived light chain of the 10E8.2.4/iMab antibody
Definition of the amino acid sequence MV 1-HC-mortar-crossover (SEQ ID NO:2) of the MV 1-derived heavy chain of the 10E8.2/iMab antibody
Definition of 10E8.2/iMab antibody 10E8.2 derived light chain amino acid sequence-10E 8.2-LC (SEQ ID NO:33)
Amino acid sequence of the 10 E8.2.3-derived heavy chain of the 10E8.2.3/iMab antibody-10 E8.2.3-HC-pestle (SEQ ID NO:39)
10E8.2.5/iMab
Amino acid sequence MV1-VLCH1(SEQ ID NO: 1) defining the MV 1-derived light chain of the 10E8.2/iMab antibody
Definition of the amino acid sequence MV 1-HC-mortar-crossover (SEQ ID NO:2) of the MV 1-derived heavy chain of the 10E8.2/iMab antibody
Definition of 10E8.2/iMab antibody 10E8.2 derived light chain amino acid sequence-10E 8.2-LC (SEQ ID NO:33)
Amino acid sequence of the 10 E8.2.5-derived heavy chain of the 10E8.2.5/iMab antibody-10 E8.2.5-HC-pestle (SEQ ID NO:40)
10E8.2.6/iMab
Amino acid sequence MV1-VLCH1(SEQ ID NO: 1) defining the MV 1-derived light chain of the 10E8.2/iMab antibody
Definition of the amino acid sequence MV 1-HC-mortar-crossover (SEQ ID NO:2) of the MV 1-derived heavy chain of the 10E8.2/iMab antibody
Definition of 10E8.2/iMab antibody 10E8.2 derived light chain amino acid sequence-10E 8.2-LC (SEQ ID NO:33)
Amino acid sequence of the 10 E8.2.6-derived heavy chain of the 10E8.2.6/iMab antibody-10 E8.2.6-HC-pestle (SEQ ID NO:41)
10E8.2.7/iMab
Amino acid sequence MV1-VLCH1(SEQ ID NO: 1) defining the MV 1-derived light chain of the 10E8.2/iMab antibody
Definition of the amino acid sequence MV 1-HC-mortar-crossover (SEQ ID NO:2) of the MV 1-derived heavy chain of the 10E8.2/iMab antibody
Definition of 10E8.2/iMab antibody 10E8.2 derived light chain amino acid sequence-10E 8.2-LC (SEQ ID NO:33)
Definition of 10E8.4/iMab antibody 10E8.4 derived heavy chain amino acid sequence-10E 8.4-HC-pestle (SEQ ID NO:42)
10E8.2.8/iMab
Amino acid sequence MV1-VLCH1(SEQ ID NO: 1) defining the MV 1-derived light chain of the 10E8.2/iMab antibody
Definition of the amino acid sequence MV 1-HC-mortar-crossover (SEQ ID NO:2) of the MV 1-derived heavy chain of the 10E8.2/iMab antibody
Definition of 10E8.2/iMab antibody 10E8.2 derived light chain amino acid sequence-10E 8.2-LC (SEQ ID NO:33)
Amino acid sequence of the 10 E8.2.8-derived heavy chain of the 10E8.2.8/iMab antibody-10 E8.2.8-HC-pestle (SEQ ID NO:43)
10E8.2.10/iMab
Amino acid sequence MV1-VLCH1(SEQ ID NO: 1) defining the MV 1-derived light chain of the 10E8.2/iMab antibody
Definition of the amino acid sequence MV 1-HC-mortar-crossover (SEQ ID NO:2) of the MV 1-derived heavy chain of the 10E8.2/iMab antibody
Definition of the amino acid sequence of the 10E 8.4-derived light chain of the 10E8.4/iMab antibody-10E 8.4-LC (SEQ ID NO:44)
Amino acid sequence of the 10 E8.2.8-derived heavy chain of the 10E8.2.8/iMab antibody-10 E8.2.8-HC-pestle (SEQ ID NO:45)
10E8.4/iMab
Amino acid sequence MV1-VLCH1(SEQ ID NO: 1) defining the MV 1-derived light chain of the 10E8.2/iMab antibody
Definition of the amino acid sequence MV 1-HC-mortar-crossover (SEQ ID NO:2) of the MV 1-derived heavy chain of the 10E8.2/iMab antibody
Definition of the amino acid sequence of the 10E 8.4-derived light chain of the 10E8.4/iMab antibody-10E 8.4-LC (SEQ ID NO:46)
Definition of 10E8.4/iMab antibody 10E8.4 derived heavy chain amino acid sequence-10E 8.4-HC-pestle (SEQ ID NO:47)
In various embodiments, at least one of the heavy chain and/or light chain sequences derived from PGT145, PG9, PGT128, PGT121, 10-1074, 3BNC117, VRC01, PGT151, 4E10, 10E8, P140, iMab (or MV1 variant), 515H7 antibody and variants thereof are paired together to form a bispecific antibody (e.g., an HIV CrossMab antibody). In an exemplary embodiment, the disclosed nucleic acid sequence is selected from the group consisting of SEQ ID NOs: 1-36 to form a bispecific antibody (e.g., an HIV CrossMab antibody).
In various embodiments, the amino acid sequence of a bispecific antibody (e.g., an HIV CrossMab antibody) also includes amino acid analogs, amino acid derivatives, or other atypical amino acids.
In various embodiments, a bispecific antibody (e.g., an HIV CrossMab antibody) comprises a sequence that is at least 60% identical to the wild-type heavy or light chain sequence of the PGT145, PG9, PGT128, PGT121, 10-1074, 3BNC117, VRC01, PGT151, 4E10, or 10E8 antibody. In various embodiments, a bispecific antibody (e.g., an HIV CrossMab antibody) comprises a sequence that is at least 60% identical to the wild-type heavy or light chain sequence of the P140, iMab (or MV1 variant), or 515H7 antibody. In exemplary embodiments, a bispecific antibody (e.g., an HIV CrossMab antibody) comprises a sequence that is at least 60% identical to any of the sequences disclosed herein.
In various embodiments, a bispecific antibody (e.g., an HIV CrossMab antibody) can comprise at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, or at least about 91% of the wild-type heavy or light chain sequence of the PGT145, PG9, PGT128, PGT121, 10-1074, 3BNC117, VRC01, PGT151, 4E10, or 10E8 antibody, A sequence that is at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical.
In various embodiments, a bispecific antibody (e.g., an HIV CrossMab antibody) can comprise at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93% >, or a combination thereof, of the wild-type heavy or light chain sequence of the P140, iMab (or MV1 variant) or 515H7 antibody, A sequence that is at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical.
In exemplary embodiments, a bispecific antibody (e.g., an HIV CrossMab antibody) can comprise at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, or a combination thereof, of any of the sequences disclosed herein, A sequence that is at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical.
Homology or identity can be determined in a variety of ways within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or megalign (dnastar) software. BLAST (basic local alignment search tool) analysis using the algorithms employed by the programs blastp, blastn, blastx, tblastn, and tblastx (Karlin et al, (1990) PROC. NATL. ACAD. SCI. USA 87, 2264. 2268; Altschul, (1993) J. MOL. EVOL.36, 290-300; Altschul et al, (1997) NUCLEIC ACIDS RES.25,3389-3402, incorporated by reference) was tailored for sequence similarity searches. The method used by the BLAST program first considers similar segments between the query sequence and the database sequences, then evaluates the statistical significance of all matches identified, and finally summarizes only those matches that meet a preselected threshold of significance. For a discussion of the basic questions regarding sequence database similarity searching, see Altschul et al, (1994)
In various embodiments, a bispecific antibody (e.g., an HIV CrossMab antibody) comprises a sequence that includes at least one amino acid alteration relative to the wild-type heavy or light chain sequence of the PGT145, PG9, PGT128, PGT121, 10-1074, 3BNC117, VRC01, PGT151, 4E10, or 10E8 antibody. In various embodiments, a bispecific antibody (e.g., an HIV CrossMab antibody) comprises a sequence that includes at least one amino acid change relative to the wild-type heavy or light chain sequence of the P140, iMab (or MV1 variant), 515H7 antibody. In exemplary embodiments, a bispecific antibody (e.g., a HIVCrossMab antibody) comprises a sequence that includes at least one amino acid change relative to any of the sequences disclosed herein.
In various embodiments, a bispecific antibody (e.g., an HIV CrossMab antibody) comprises an amino acid sequence that includes at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 40, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 74, 76, 72, 76, 77, 76, 75, 77, 79, or 79 amino acid alterations relative to the wild-type heavy or light chain sequence of the PGT145, PG9, PGT128, PGT121, 10-1074, 3BNC117, VRC01, PGT151, 4E10, or 10E8 antibody.
In various embodiments, a bispecific antibody (e.g., an HIV CrossMab antibody) comprises a sequence that includes at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 40, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 amino acid changes relative to the wild-type heavy or light chain sequence of a P140, iMab (or MV1 variant) or 515H7 antibody.
In exemplary embodiments, a bispecific antibody (e.g., an HIV CrossMab antibody) comprises a sequence that includes at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 40, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 amino acid changes relative to any of the sequences disclosed herein.
The amino acid change may be an amino acid deletion, insertion, substitution or modification. In one embodiment, the amino acid change is an amino acid deletion. In another embodiment, the amino acid change is an amino acid substitution.
In various embodiments, the amino acid change can be in a Complementarity Determining Region (CDR) of the bispecific antibody (e.g., a CDR1, a CDR2, or a CDR3 region). In another embodiment, the amino acid change can be in a framework region (FW) of the bispecific antibody (e.g., FW1, FW2, FW3, or FW4 region). In yet another embodiment, the amino acid change can be in a junction region (J region) of the bispecific antibody (e.g., J1, J2, J3, J4, J5, J6, or J7 regions).
Also provided herein are chimeric antibody derivatives of bispecific antibodies, i.e., antibody molecules in which a portion of the heavy and/or light chain is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remaining portion of the chain is identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity. For example, a bispecific antibody may comprise a heavy chain and/or a light chain in which one or more CDRs or FWs derived from an antibody selected from the group consisting of PGT145, PG9, PGT128, PGT121, 10-1074, 3BNC117, VRC01, PGT151, 4E10, 10E8, P140, iMab (or MV1 variant) or 515H7 antibody are substituted with one or more CDRs or FWs derived from a different antibody selected from the group consisting of PGT145, PG9, PGT128, PGT121, 10-1074, 3BNC117, VRC01, PGT151, 4E10, 10E8, P140, iMab (or MV1 variant) or 515H7 antibody.
In various embodiments, the present invention provides improved bispecific antibodies that exhibit advantageous properties with respect to solubility, stability, and therapeutic activity. It is envisaged that such antibodies may be particularly suitable for large scale commercial production. For example, such antibodies may exhibit increased solubility, reduced aggregation, reduced precipitation, and/or increased stability or resistance to degradation during manufacture.
In an exemplary embodiment, the improved bispecific antibody is a variant of the 10E8V2.0/iMab antibody (also referred to as the 10E8.2/iMab antibody). In such embodiments, the variant may exhibit increased solubility, stability, and/or therapeutic activity (e.g., antiviral activity) as compared to the parent 10E8V2.0/iMab antibody.
In some embodiments, a bispecific antibody (e.g., an HIV Crossmab antibody) may comprise at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89% >, or a combination thereof, of the heavy or light chain sequences of the 10E8V2.0/iMab antibody (i.e., SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:33, or SEQ ID NO:34), A sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical.
In various embodiments, a bispecific antibody (e.g., an HIV Crossmab antibody) may comprise a heavy chain or light chain sequence comprising at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 relative to the heavy or light chain sequence of the 10E8V2.0/iMab antibody (i.e., SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:33, or SEQ ID NO:34), 41. 42, 43, 44, 45, 46, 47, 40, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79 or 80 amino acid changes.
In various embodiments, the bispecific antibody may comprise one or more amino acid changes in the Complementarity Determining Regions (CDRs) of the 10E8V2.0/iMab antibody (e.g., CDR1, CDR2, or CDR3 regions). In another embodiment, the bispecific antibody can comprise one or more amino acid alterations in the framework regions (FWs) of the bispecific antibody (e.g., the FW1, FW2, FW3, or FW4 regions). In yet another embodiment, the amino acid change may be in the junction region (J region) of the 10E8V2.0/iMab antibody (e.g., J1, J2, J3, J4, J5, J6, or J7 region).
In some embodiments, the bispecific antibody comprises a variant heavy chain derived from 10E8V2.0 (i.e., SEQ ID NO: 34). In such embodiments, the bispecific antibody may comprise one or more mutations at a position selected from L72, I75, F77, L89, Y98, F100a, W100b, Y100e, P100F, P100g, L108 and/or L170 of the heavy chain (the mutation position on seq id NO:34 is determined by the Kabat numbering system). In some embodiments, the bispecific antibody may comprise one or more mutations at positions selected from L72, I75, F77, and/or L108. In some embodiments, the bispecific antibody may comprise one or more mutations selected from L72K, I75K, F77T, and L108K. In one embodiment, the bispecific antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 47.
In some embodiments, the bispecific antibody comprises a variant light chain derived from 10E8V2.0 (i.e., SEQ ID NO: 33). In such embodiments, the bispecific antibody may comprise one or more mutations at a position selected from the group consisting of L15, P40, I45 and P112 of the light chain (the position of the mutation on SEQ ID NO:33 is determined by the Kabat numbering system). In some embodiments, the bispecific antibody may comprise one or more mutations at a position selected from P40 and I45. In some embodiments, the bispecific antibody may comprise one or more mutations selected from P40T and I45K. In one embodiment, the bispecific antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 46.
In some embodiments, the bispecific antibody comprises a variant light chain derived from MV1 (i.e., SEQ ID NO: 1). In such embodiments, the bispecific antibody may comprise mutations at positions 52-54 (the positions of the mutations in SEQ ID NO:1 are determined by the Kabat numbering system). In some embodiments, the bispecific antibody may comprise amino acid mutations at positions 52-54. In some embodiments, the amino acid mutation at position 52 is asn (n), the amino acid mutation at position 53 is ser(s), and the amino acid mutation at position 54 is thr (t). In some embodiments, the Asn mutation at position 52 is N-linked glycosylated. In one embodiment, the bispecific antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 38.
In an illustrative embodiment, the bispecific antibody comprises a heavy chain and a light chain derived from 10E8 comprising the amino acid sequences SEQ ID NO:47 and SEQ ID NO: 46. The bispecific antibody further comprises a heavy chain and a light chain derived from MV1, said heavy chain and light chain comprising the amino acid sequences SEQ ID NO:1 and
Modification of the amino acid sequence of recombinant binding proteins is accomplished using any technique known in the art, such as site-directed mutagenesis or PCR-based mutagenesis. Such techniques are described, for example, in Sambrook et al, Molecular Cloning, analytical Manual, Cold Spring Harbor Press, Plainview, N.Y.,1989, and Ausubel et al, Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y., 1989.
Methods for producing antibodies such as those disclosed herein are known in the art. For example, DNA molecules encoding the light chain variable region and/or the heavy chain variable region can be chemically synthesized using the sequence information provided herein. The synthetic DNA molecule may be linked to other appropriate nucleotide sequences, including, for example, expression control sequences, to produce conventional gene expression constructs encoding the desired antibody. The generation of the defined gene constructs is within the routine skill in the art. Alternatively, the sequences provided herein can be cloned from a hybridoma by conventional hybridization techniques or Polymerase Chain Reaction (PCR) techniques using synthetic nucleic acid probes whose sequences are based on the sequence information provided herein or prior art sequence information for the genes encoding the heavy and light chains.
The nucleic acid encoding the desired antibody may be incorporated (ligated) into an expression vector, which may be introduced into the host cell by conventional transfection or transformation techniques. Exemplary host cells are E.coli cells, Chinese Hamster Ovary (CHO) cells, human embryonic kidney 293(HEK 293) cells, HeLa cells, Baby Hamster Kidney (BHK) cells, monkey kidney Cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), and myeloma cells that do not otherwise produce IgG proteins. The transformed host cell may be grown under conditions that allow the host cell to express the genes encoding the immunoglobulin light and/or heavy chain variable regions. The particular expression and purification conditions will vary depending on the expression system used.
In various embodiments, bispecific antibodies of the invention (e.g., HIV CrossMab antibodies) are used in therapy. For example, bispecific antibodies (e.g., HIV CrossMab antibodies) can be used to neutralize HIV in a mammal (e.g., a human patient). For example, the antibodies of the invention may bind to HIV so as to partially or completely inhibit one or more biological activities of the virus. In one embodiment, the bispecific antibody (e.g., an HIV CrossMab antibody) neutralizes R5 tropic HIV. In another embodiment, the bispecific antibody (e.g., an HIV CrossMab antibody) neutralizes X4 tropic HIV. In yet another embodiment, the bispecific antibody (e.g., an HIV CrossMab antibody) neutralizes R5X4 amphotropic HIV. In some embodiments, using an antibody to neutralize HIV in a mammal comprises administering to the mammal a therapeutically effective amount of the antibody.
Typically, a therapeutically effective amount of the active ingredient is in the range of, for example, about 0.1mg/kg to about 100mg/kg, such as about 1mg/kg to about 10mg/kg of the patient's body weight. In various embodiments, a therapeutically effective amount of the active component is in the range of about 0.01mg/kg to about 30mg/kg of patient body weight, e.g., about 0.01mg/kg, about 0.02mg/kg, about 0.03mg/kg, about 0.04mg/kg, about 0.05mg/kg, about 0.06mg/kg, about 0.07mg/kg, about 0.08mg/kg, about 0.09mg/kg, about 0.1mg/kg, about 0.2mg/kg, about 0.3mg/kg, about 0.4mg/kg, about 0.5mg/kg, about 0.6mg/kg, about 0.7mg/kg, about 0.8mg/kg, about 0.9mg/kg, about 1mg/kg, about 1.1mg/kg, about 1.2mg/kg, about 1.3mg/kg, about 1.4mg/kg, about 1.5mg/kg, about 1.6mg/kg, about 1.1mg/kg, about 1.8mg/kg, about 1.6mg/kg, about 1.1mg/kg, about 1.4mg/kg, about 1.6mg/, 1.9mg/kg, about 2mg/kg, about 3mg/kg, about 4mg/kg, about 5mg/kg, about 6mg/kg, about 7mg/kg, about 8mg/kg, about 9mg/kg, about 10mg/kg, about 11mg/kg, about 12mg/kg, about 13mg/kg, about 14mg/kg, about 15mg/kg, about 16mg/kg, about 17mg/kg, about 18mg/kg, about 19mg/kg, about 20mg/kg, about 21mg/kg, about 22mg/kg, about 23mg/kg, about 24mg/kg, about 25mg/kg, about 26mg/kg, about 27mg/kg, about 28mg/kg, about 29mg/kg, about 30mg/kg body weight, including all values and ranges therebetween.
In some embodiments, a therapeutically effective amount of an active component is any value between about 1mg/kg to 10mg/kg, between about 10mg/kg to 20mg/kg, between about 20mg/kg to 30mg/kg, between about 30mg/kg to 40mg/kg, between about 40mg/kg to 50mg/kg, between about 50mg/kg to 60mg/kg, between about 60mg/kg to 70mg/kg, between about 70mg/kg to 80mg/kg, between about 80mg/kg to 90mg/kg, or between about 90mg/kg to 100 mg/kg.
In some embodiments, a therapeutically effective amount of the active component is about 1mg/kg, about 2mg/kg, about 3mg/kg, about 6mg/kg, about 10mg/kg, about 20mg/kg, about 30mg/kg, or about 60mg/kg of Intravenous (IV) delivery.
In some embodiments, a therapeutically effective amount of the active component is about 2.5mg/kg, about 5mg/kg, about 10mg/kg, or about 20mg/kg delivered subcutaneously (s.c.) or intramuscularly (i.m).
The amount administered will depend on variables such as the type and extent of the disease or indication to be treated, the overall health of the patient, the in vivo efficacy of the antibody, the pharmaceutical formulation and the route of administration. The initial dose may be increased beyond the upper limit in order to quickly achieve the desired blood or tissue level. Alternatively, the initial dose may be less than optimal and the dose may be escalated during the course of treatment. Human doses may be optimized, for example, in a conventional phase I dose escalation study designed to increase from, for example, 0.5mg/kg to 20 mg/kg. The frequency of administration may vary depending on factors such as the route of administration, the dosage and the condition being treated. Exemplary dosing frequencies are more than once per day, about twice per day, about three times per day, about four times per day, about five times per day, about every other day, about every three days, about once per week, about once per two weeks, about once per month, about once per two months, about once per three months, about once per six months, or about once per year. Formulation of antibody-based drugs is within the ordinary skill in the art.
In various embodiments, the antibodies of the invention can be administered chronically. For example, the antibody may be administered for at least about 1 week, at least about 4 weeks, about 8 weeks, or at least about 12 weeks. In some embodiments, the regimen lasts at least about 1 month, at least about 6 months, at least about 12 months, at least about 1 year, at least about 2 years, at least about 3 years, at least about 4 years, at least about 5 years, at least about 6 years, at least about 7 years, at least about 8 years, at least about 9 years, at least about 10 years, at least about 15 years, at least about 20 years, at least about 30 years, at least about 40 years, or at least about 50 years.
For therapeutic use, the antibody may be combined with a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" refers to buffers, carriers, and excipients that are suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. The carrier should be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. Pharmaceutically acceptable carriers include buffers, solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is known in the art.
Pharmaceutical compositions containing antibodies such as those disclosed herein can be presented in dosage unit form and can be prepared by any suitable method. The pharmaceutical composition should be formulated to be compatible with its intended route of administration. Examples of routes of administration are Intravenous (IV), intradermal, inhalation, transdermal, topical, transmucosal and rectal administration. In one embodiment, the route of administration of the antibody of the invention is IV infusion. Useful formulations may be prepared by methods well known in the pharmaceutical arts. See, for example, Remington's Pharmaceutical Sciences, 18 th edition (Mack Publishing Company, 1990).
In some embodiments, the pharmaceutical composition is formulated as a composition suitable for oral administration. Compositions for oral delivery may be in the form of, for example, tablets, lozenges, aqueous or oily suspensions, granules, powders, emulsions, capsules, syrups or elixirs. Compositions for oral administration may comprise one or more agents, for example sweetening agents such as fructose, aspartame or saccharin; flavoring agents, such as peppermint, oil of wintergreen, or cherry red; a colorant; and preservatives to provide pharmaceutically palatable preparations.
In some embodiments, the pharmaceutical composition is formulated as a composition suitable for parenteral administration. Dosage forms suitable for parenteral administration (e.g., intravenous, subcutaneous, intramuscular, or intraperitoneal injection and infusion) include, for example, solutions, suspensions, dispersions, emulsions, and the like. They may also be manufactured in the form of sterile solid compositions (e.g., lyophilized compositions), which may be dissolved or suspended in a sterile injectable medium just prior to use. They may contain, for example, suspending or dispersing agents.
In some embodiments, the composition may additionally comprise a pharmaceutically acceptable excipient or carrier. Exemplary excipients include sodium citrate, dicalcium phosphate and the like, and/or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, silicic acid, microcrystalline cellulose, and baker's Special Sugar (Bakers Special Sugar) and the like, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, acacia, polyvinyl alcohol, polyvinylpolypyrrolidone, methylcellulose, Hydroxypropylcellulose (HPC), and hydroxymethylcellulose and the like, c) humectants such as glycerol and the like, d) disintegrating agents such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, sodium carbonate, cross-linked polymers such as crospovidone (cross-linked polyvinylpyrrolidone), cross-linked carboxymethylcellulose sodium (cross-linked sodium carboxymethylcellulose), sodium starch glycolate and the like, e) solution retarding agents, such as paraffin and the like, f) absorption promoters, such as quaternary ammonium compounds and the like, g) wetting agents, such as, for example, cetyl alcohol and glyceryl monostearate and the like, h) absorbents, such as kaolin and bentonite and the like, and i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, glyceryl behenate and the like, and mixtures of such excipients. One skilled in the art will recognize that a particular excipient may have two or more functions.
The pharmaceutical formulation is preferably sterile. Sterilization may be achieved, for example, by filtration through sterile filtration membranes. In the case of a composition that is lyophilized, it may be filter sterilized before or after lyophilization and reconstitution.
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