KR20210106001A - mRNA Display Antibody Libraries and Methods - Google Patents

Abstract

Compositions, methods and uses of recombinant viruses and/or recombinant viral vectors encoding distinct antibodies or antibody fragments generated from high diversity nucleic acid libraries are provided. Preferably, the recombinant virus is a genetically modified low immunogenicity virus, such as an E2b-deleting adenovirus. The high diversity nucleic acid library comprises or is _{derived from (1) a V H} -CDR1/2 sub-library, (2) a plurality of V _H -CDR3 sub-libraries, and (3) a V _L sub-library, each of which It contains multiple members. Preferably, each member of the sub-library comprises at least one random cassette with a plurality of degenerate base positions. In a particularly preferred embodiment _{, at least a portion of at least two members of the V H} -CDR1/2 sub-library, the plurality of V _H -CDR3 sub-libraries and the V _L sub-library are recombined to form an expression library member in the expression library. and each member of the expression library encodes a separate antibody or antibody fragment.

Description

mRNA Display Antibody Libraries and Methods

The field of the present invention relates to compositions and methods for ultrahigh-diversity antibody libraries, in particular to the use of mRNA display libraries and mRNA display libraries for generating recombinant high-affinity binding agents.

The background description contains information that may be useful in understanding the present invention. It is not an admission that any information provided herein is prior art or is related to the invention claimed herein, or that any publication specifically or implicitly recited is prior art.

All publications and patent applications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. To the extent a definition or use of a term in an incorporated reference is inconsistent or contradictory with the definition of the term provided herein, the definition of the term provided herein applies and the definition of the term in the reference does not apply.

Targeting tumor antigens or neoepitopes using specific antibodies or binding molecules with high affinity has proven to be an effective method for treating cancer patients. As more and more patient-specific and/or cancer-specific tumor antigens and/or neoepitopes are identified in vivo, in vitro or in silico through omics data analysis, stable, There has been a growing need to generate antibody libraries or display libraries that are soluble, functional, and have a high potential for selection of adaptable antibodies or binding agents. A specific antibody or binding molecule of high affinity may be identified or derived from a pool of natural antibodies, but the identified or derived native antibody or binding agent may depend on the frequency or intensity of exposure to the antigen or neoepitope. It may or may not be effective as the diversity of antibodies may be limited.

In one approach to solving the above problem, a recombinant phage display library can be used. Although this approach allows the generation of libraries with reasonably high diversity, many rounds of enrichment for binding agents are often required, which is labor intensive and time consuming. Also, despite their relatively large diversity, binders tend to have less than ideal affinity and stability. Furthermore, diversity is typically limited by practical considerations such as library volume, transfection efficiency, and the like. This and other approaches can be further optimized using multiple artificial selective pressures, for example as described in WO 2006/072773. Although this method can improve stability characteristics, it requires a significant amount of library manipulation and time.

In another approach, mRNA display can be performed. wherein an mRNA sequence encoding a candidate binding molecule (typically a scFv) is coupled at its 3′-end with a puromycin molecule, and a peptide encoded by the mRNA sequence is generated via in vitro translation, converting the mRNA into mRNA to generate a fusion product coupled directly to the protein encoded by However, current mRNA display technologies advantageously avoid problems associated with transfection limitations and allow at least conceptually higher diversity, but problems with structural integrity or stability, relatively low affinity and/or cross-reactivity. is still left To further improve at least selected binding characteristics of the scFv from mRNA display, a VH-CDR3 spectratyping analysis was performed ( Protein Engineering, Design & Selection , 2015, vol. 28 no. 10, pp. 427). -435]). However, this process requires repeat analysis and may not be productive for all antigens.

Thus, while methods for generating and identifying candidate binders using mRNA display and other methods are known, high diversity libraries with binders with high structural integrity/stability, low affinity and/or low cross-reactivity are difficult to achieve. remains difficult. Accordingly, there is still a need for improved compositions, methods and uses of mRNA display for the rapid production of stable recombinant high affinity binders.

The subject of the present invention is a high diversity nucleic acid library encoding a plurality of antibodies or antibody fragments allowing reliable and efficient identification of stable, soluble and functional antibodies or binding agents against various biomolecules, in particular cancer antigens or neoepitopes. to various compositions, methods therefor and uses thereof. Accordingly, one aspect of the above subject matter includes methods for generating a high diversity nucleic acid library encoding a plurality of antibodies or antibody fragments. In this method, three sub-libraries each having a plurality of members: (1) V _H -CDR1/2 sub-library, (2) a plurality of V _H -CDR3 sub-libraries, and (3) V _L sub-library is created or provided. Each member of the three sub-libraries comprises at least one random cassette with a plurality of degenerate base positions. At least a portion of at least two members of the three libraries recombine to form an expression library member in the expression library, which has a plurality of expression library members. Each expression library member encoding a separate antibody or antibody fragment. In a preferred embodiment, the expression library member is transcribed into an mRNA fragment, which is then coupled at the 3'-end with a puromycin molecule.

In another aspect of the present subject matter, the inventors contemplate compositions having a plurality of nucleic acid libraries. The plurality of nucleic acid libraries comprises (1) a V _H -CDR1/2 sub-library, (2) a plurality of V _H -CDR3 sub-libraries, and (3) a V _L sub-library. Each of the sub-libraries (1) to (3) comprises a plurality of members, and each member of the sub-library comprises at least one random cassette having a plurality of degenerate base positions.

In another aspect of the subject matter of the present invention, the present inventors contemplate the use of said composition for generating a high diversity nucleic acid library.

In another aspect of the present subject matter, the present inventors contemplate high diversity nucleic acid library compositions having a plurality of library members. A high diversity nucleic acid library member comprises a recombinant nucleic acid comprising a plurality of random cassettes each having a plurality of degenerate base positions. The plurality of random cassettes comprises at least _{2 from (1) a V H} -CDR1/2 sub-library, (2) a plurality of V _H -CDR3 sub-libraries and (3) any two libraries from the _{V L sub-library.} derived from members of the dog.

In another aspect of the present subject matter, the present inventors contemplate the use of a high diversity nucleic acid library for generating therapeutic recombinant antibodies to cancer neoepitopes.

In another aspect of the present subject matter, the present inventors contemplate methods of producing recombinant antibodies. In this method, three sub-libraries each having a plurality of members: (1) V _H -CDR1/2 sub-library, (2) a plurality of V _H -CDR3 sub-libraries, and (3) V _L sub-library is created or provided. Each member of the three sub-libraries comprises at least one random cassette with a plurality of degenerate base positions. At least a portion of at least two members of the three libraries recombine to form an expression library member in the expression library, which has a plurality of expression library members. Each expression library member encoding a separate antibody or antibody fragment. Thereafter, the method continues to produce recombinant antibodies or fragments thereof using expression library members.

In another aspect of the present subject matter, the present inventors contemplate a method of isolating a high affinity binding agent having an affinity of 100 nM or less for an antigen by contacting the antigen with a composition constructed by the method described above.

In another aspect of the present subject matter, the inventors contemplate recombinant nucleic acid fragments produced using oligonucleotides selected from Table 1 or Table 2 provided below.

In another aspect of the present subject matter, the inventors contemplate a synthetic nucleic acid mixture having a nucleic acid sequence selected from Table 1 or Table 2 provided below.

In another aspect of the subject matter of the present invention, we contemplate a recombinant virus. Recombinant viruses include recombinant nucleic acids comprising members of expression libraries encoding distinct antibodies or antibody fragments. A member of the expression library _{generates or provides (1) a V H} -CDR1/2 sub-library, (2) a plurality of V _H -CDR3 sub-libraries and (3) a V _L sub-library, wherein the sub-library (1) to (3) each comprises a plurality of members, wherein each member of the sub-library comprises at least one random cassette having a plurality of degenerate base positions), V _H -CDR1/2 sub -recombining at least a portion of at least two members of the library, the plurality of V _H -CDR3 sub-libraries and the V _L sub-library to form an expression library member in the expression library.

Typically, the recombinant virus is a genetically modified low immunogenicity virus, which may most preferably be human adenovirus serotype 5 having a mutation in at least one of genes E1A, E1B, E2B, E3.

In some embodiments, the plurality of members of the _{V H} -CDR1/2 sub-library comprises a random cassette corresponding to at least one of a portion of a _{V H} CDR1 and a portion of a V _{H CDR2.} In this embodiment, the plurality of members of the _{V H} -CDR1/2 sub-library comprises a plurality of random cassettes corresponding to at least a portion of the _{V H CDR2.} In other embodiments, the plurality of members of the _{V H} -CDR1/2 sub-library comprises a plurality of random cassettes corresponding to _{at least a portion of the V H} CDR1 and a portion of the V _{H CDR2.}

In some embodiments, the plurality of members of the _{V H} -CDR3 sub-library comprises a random cassette corresponding to at least a portion of the _{V H CDR3.} V _H -CDR3 sub-random at least two cassettes of a member of the library is considered to encode peptides having different lengths. Alternatively and/or additionally, the plurality of members of the _{V L} sub-library comprises a random cassette in a portion of the _{V L CDR3.}

Typically, the recombination step is V _H -CDR1 / 2 sub-library and a plurality of V _H -CDR3 sub-separating at least a portion of one member of the library, and, by combining them with V _H domains in the V _H domain library library members _{forming a V H} domain library, wherein the V H domain library comprises a plurality of V _H domain library members. In this embodiment, the members of the expression library is a V _L sub-separating at least a portion of a member of the library, and, V _L sub-fusing one of the part of the members of the library and the V _H domain library member by forming the expression library members considered to be generated. In another embodiment, the recombination step _{isolates at least a portion of the members of one of the V H} -CDR1/2 sub-library and the plurality of V _H -CDR3 sub-libraries and fuses them together to obtain a first group of expression library members. includes forming.

Optionally, the recombinant nucleic acid may further comprise a nucleic acid fragment encoding a signaling peptide that promotes secretion of a separate antibody or antibody fragment.

In another aspect of the present subject matter, the present inventors contemplate methods of producing recombinant antibodies. In this method, (1) a V _H -CDR1/2 sub-library, (2) a plurality of V _H -CDR3 sub-libraries and (3) a V _L sub-library are created or provided, the sub-library (1) ) to (3) each comprises a plurality of members. Most preferably, each member of the sub-library comprises at least one random cassette having a plurality of degenerate base positions. Thereafter, the method comprises _{recombination of at least a portion of at least two members of a V H} -CDR1/2 sub-library, a plurality of V _H -CDR3 sub-libraries and a V _L sub-library to form an expression library member in the expression library. Continuing with step, the expression library comprises a plurality of expression library members, each expression library member encoding a separate antibody or antibody fragment. Recombinant viral vectors can then be generated comprising at least one expression library member.

Typically, the recombinant virus is a genetically modified low immunogenicity virus, which may most preferably be human adenovirus serotype 5 having a mutation in at least one of genes E1A, E1B, E2B, E3.

In some embodiments, the random cassette is generated using an oligonucleotide selected from SEQ ID NO:1 to SEQ ID NO:25. Optionally, the recombinant viral vector further comprises a nucleic acid fragment encoding a signaling peptide that promotes secretion of a separate antibody or antibody fragment.

Preferably, the method may further comprise contacting the recombinant virus carrying the recombinant viral vector with the mammalian cell. In some embodiments, the contacting step comprises administering the recombinant virus to the mammal. In another embodiment, the mammalian cells are autologous cells of a patient having a tumor, and the contacting comprises co-incubating the autologous cells with the mammalian cells ex vivo.

Various objects, features, aspects and advantages of the present subject matter will become more apparent from the following detailed description of preferred embodiments taken in conjunction with the accompanying drawings.

1 illustrates one exemplary randomization strategy using VH3/Vk1 pairs.
2 illustrates exemplary positions for sequence randomization in heavy chain CDR1 and CDR2.
3 illustrates exemplary sequence randomization in heavy chain CDR3.
4 illustrates exemplary sequence randomization in light chain CDR3 with nucleic acid sequence on the left and amino acid selection on the right.
5 illustrates exemplary production of hybrid nucleic acid elements by isolating and combining random cassettes of multiple recombinant nucleic acid segments.
6 presents the results of size exclusion chromatography showing a single peak indicating stable protein expression of αB7-H4 _801.
7 presents capillary electrophoresis sodium dodecyl sulfate (CE-SDS) data showing similar molecular behavior of _{αB7-H4 801} compared to commercial antibody.
8 presents a graph showing the binding of selected αB7-H4 antibodies to B7-H4 in vitro.
9 presents a graph of functional analysis of selected αB7-H4 binding agents and αPD-L1 binding agents in vitro.
10 presents a graph showing the binding affinities of αB7-H4 scFv and αB7-H4 IgG1.
11 presents an IL-8 activity assay and its results by measuring changes in neutrophil size.
12 shows a bar graph showing the neutralizing effect of αIL-8 antibody on IL-8 activity of increasing neutrophil size.
13 shows the IL-8 activity assay and its results presented in a bar graph showing the neutralizing effect of αIL-8 antibody on IL-8 activity by inhibiting neutrophil migration.
14 shows exemplary results using the mRNA display library compositions presented herein with respect to selected antigenic targets.
15 presents an exemplary graph depicting the affinity of selected binding agents comprised of scFv versus IgG, wherein binding agents were identified using the mRNA display library compositions presented herein.

The present inventors now propose that specific and effective recombinant antibodies or fragments thereof can be generated or identified by constructing high diversity nucleic acid libraries using targeted diversification of selected domains of antibodies or fragments thereof encoded by members of the high diversity nucleic acid library. found that there is To achieve the above object, the present inventors now propose that one or more domains or subdomains of an antibody/binding agent can be preselected, and a plurality of nucleic acid sub-libraries can be generated using random cassettes in the preselected domains or subdomains. found that there is The present inventors have found that members of sub-libraries are recombine to allow for high diversity among library members, while for use in vivo against cancer antigens or neoepitopes (preferably cancer-specific, patient-specific neoepitopes or neoantigens). It has further been discovered that high diversity nucleic acid libraries can be constructed that provide a higher possibility of identifying stable, soluble, functional, and adaptable antibodies/binding agents when provided.

Indeed, and as will be shown in more detail below, the libraries presented herein allow the isolation of at least one binding agent to any antigen, typically in single or two-pass enrichment, wherein the binding agent is less than or equal to 100 nM, more It typically has _{a K d} of 10 nM or less. Also contemplated systems and methods are that scFv libraries are all at least 10 ⁹ , at least 10 ¹⁰ , at least 10 ¹¹ , at least 10 ¹² , at least 10 ¹³ , at least 10 ¹⁴ , at least 10 within a significantly reduced period compared to conventional library constructs. ^It is possible to have a diversity of 15 or at least 10 ^{16 distinct library members.} Accordingly, it should be appreciated that the rate of antibody discovery is substantially increased.

As used herein, the term “tumor” refers to and is used interchangeably with one or more cancer cells, cancer tissues, malignant tumor cells, or malignant tumor tissues that may be present or found at one or more anatomical locations in the human body.

As used herein, the term “binds” refers _{to an interaction between two molecules with high affinity having a K D} ^{of 10 −6} M or less or 10 ⁻⁷ M or less, and the terms “recognize” and/or “ can be used interchangeably with "to detect".

As used herein, the term "provide" or "providing" refers to and includes any act of making, producing, disposing, making available, or preparing for use.

Construction of Nucleic Acid Sub-Libraries

In general, the structural components of an antibody (heavy chain, light chain, constant domain, variable domain) are closely related to its function. For example, the variable domain of the heavy chain (V _H) and light chain (V _L) is configured with an epitope-binding domain that provides specificity to the antibody. Each of V _H and V _L comprises three complementarity determining regions (CDRs, CDRs 1 to 3) with unique amino acid sequences based on specificity for the antigen. Thus, it was previously contemplated that recombinant nucleic acid libraries for generating or identifying antibodies can be generated by randomizing the sequences encoding the CDRs of _{V H} and V _{L .} However, we have found _{that while full randomization of all CDRs of V H} and V _L can provide great diversity to the library, all randomized V _H and V _L recombine to form antibodies (eg, IgG1, etc.) was found to also create inefficiencies in generating all combinations of random sequences and screening all randomized combinations as they may not be soluble or stably expressed when In addition, it is not practical to cover the entire diversity space due to the very large number of possible library members.

Thus, the inventors of the present invention include, for general common framework region and the final peptide product (for example, between the V _H or V _L V a sub-domain of the _H and V _L different antibodies (or genes encoding the antibody), scFv, It is contemplated that it can be divided into two classes of targeted diversification regions that can be at least partially or fully randomized without significantly affecting the stability and/or solubility of IgG1, etc.). _{Preferably, the targeted diversification region of V H} comprises at least a portion of CDR1, CDR2-n (N-terminal side of CDR2), CDR2-c (C-terminal side of CDR2) and CDR3. In a further preferred embodiment, _{the targeted diversification region of V L} comprises at least a portion of CDR3.

As such, in one exemplary and particularly preferred embodiment of the present subject matter, a nucleic acid library is generated by generating recombinant nucleic acids comprising one or more random sequence cassettes in one or more targeted diversification regions of _{V H} and/or V _{L .} can be In one preferred embodiment, we randomize, avoiding too many randomized recombination sequences in a single sub-library, where the volume of a single sub-library can make it impractical or inefficient to handle rapid or adequate screening. Consider three different sub-libraries with different sets of random sequence cassettes within different targeted diversification regions such that each sub-library possesses diversity within the targeted targeted diversification region. In addition, conserved regions between targeted diversification regions are selected or designed for maximum stability and solubility.

In one embodiment, the sub-library comprises a V _H -CDR1/2 sub-library. A V _H -CDR1/2 sub-library comprises a plurality of recombinant nucleic acids (eg, recombinant DNA) having one or more random sequence cassettes corresponding to at least a portion _{of a V H CDR1 and/or a portion of a V H CDR2.} As used herein, the random cassette corresponding to the portion of the V _H CDR1 is random cassette and means located in the area on the recombinant nucleic acid, wherein the sequence encoding the CDR1 portion is at least structurally, or the V _H domain of a natural antibody It must exist to encode a portion of a functionally similar V _{H domain.} For example, _{the recombinant nucleic acid of the V H} -CDR1/2 sub-library may have the following structure (randomized regions are underlined and fixed sequence regions are in parentheses):

5'- (promoter -5' UTR - FW1) + CDR1 + (FW2) + CDR2 + (FW3 - CDR3 - FW4)

As used herein, UTR refers to untranslated region and FW refers to framework region (eg, FW1 is a first framework region distinguishable from a second framework region FW2). In this structure, a random sequence cassette may be inserted in the region of CDR1 or CDR2, or preferably both CDR1 and CDR2. In some embodiments, more than one random sequence cassette, preferably two random sequence cassettes, may be inserted in the region of CDR2: CDR2-n (5'-terminal side of CDR2) and CDR-c (3 of CDR2) '-end side).

The sub-library may also include a plurality of V _H -CDR3 sub-libraries. Each sub-library of V _H -CDR3 comprises a plurality of recombinant nucleic acids (eg, recombinant DNA) having one or more random sequence cassettes corresponding to at least a portion of the _{V H CDR3.} V _H -CDR1 / 2 sub-library analogy, V _H -CDR1 / 2 sub-library of recombinant nucleic acid may have a structure as described below (randomized regions are underlined, fixed sequence regions parentheses exists in):

5'- (promoter - 5' UTR - FW1 + CDR1 + FW2 + CDR2 + FW3) - CDR3 - (FW4)

Preferably, _{the fixed sequence of the recombinant nucleic acid of the V H} -CDR1/2 sub-library and/or the V _H -CDR3 sub-library (eg promoter - 5' UTR - FW1 + CDR1 + FW2 + CDR2 + FW3) , FW4) are native antibodies such that the immobilized sequence is most expressible and adaptable to a number of formats, including single chain variable fragments (scFv), modified forms of scFv, full-length immunoglobulins or peptides expressed as part of an immunoglobulin. (eg, IgG1 to various antigens) are selected to use the most common and/or conserved sequences. Thus, in a preferred embodiment, _{the immobilized sequences of the recombinant nucleic acid of the V H} -CDR1/2 sub-library and the recombinant nucleic acid of the V _H -CDR3 sub-library are at least 70%, preferably at least 80%, of each other, more preferably are at least 90% identical (shared).

A sub-library may also include a V _L sub-library. V _L sub-library includes a plurality of recombinant nucleic acid having at least one random sequence cassette corresponding to at least a portion of the V _L CDR3 comprises (e.g., recombinant DNA). V _H -CDR1 / 2 sub-library analogy, V _H -CDR1 / 2 sub-library of recombinant nucleic acid may have a structure as described below (randomized regions are underlined, fixed sequence regions parentheses exists in):

5'- (promoter - 5' UTR - FW1 + CDR1 + FW2 + CDR2 + FW3) - CDR3 - (FW4)

Preferably, the immobilized sequence of the recombinant nucleic acid of the _{V L} sub-library is at least 70%, preferably at least 80%, that of the recombinant nucleic acid of the _{V H} -CDR1/2 sub-library or the V _{H -CDR3 sub-library,} more preferably at least 90% identical (shared).

Although any randomization sequence can be considered to generate a random sequence cassette, the inventors have found that a stratified random sequence cassette for the CDR1, CDR2, CDR3 _{of V H and CDR3 of the V L} domain can be used for binding peptides (e.g., scFv, etc.) are considered to provide a binding surface of high complexity and great potential. For example, the _{stratified random sequence cassette for CDR1, CDR2 of the V H} -CDR1/2 sub-library may contain no more than 3, preferably no more than 2, more preferably no more than 1 random sequence per cassette (3 per cassette). encoding no more than 2, 2 or 1 random amino acid). The position of a random sequence in a random cassette may vary depending on the random amino acid within the cassette. In another example, the _{stratified random sequence cassette for CDR3 of the V H} -CDR3 sub-library contains 4 or more, preferably 5 or more, more preferably 6 or more random sequences per cassette (4 or more per cassette). , preferably encoding at least 5 or more preferably at least 6 random amino acids). In another example, _{the stratified random sequence cassette for CDR3 of the VL} sub-library contains at least 4, preferably at least 5, more preferably at least 6 random sequences per cassette (at least 4, preferably at least 4 per cassette). (encoding at least 5 or more preferably at least 6 random amino acids).

In a particularly preferred embodiment of the present subject matter, the inventors have determined that preferred random sequence cassettes for sub-libraries are _{listed in Table 1 (V H} -CDR1/2 sub-library and V _H -CDR3 sub-library) and Table 2 (V _L sub-library) can be generated using the oligonucleotides presented in As shown in Tables 1 and 2 , each oligonucleotide comprises a random sequence (highlighted) with a degenerate code represented by the IUPAC ambiguity code. For example, one oligonucleotide for the CDR1 random sequence cassette comprises a random sequence "RVT" representing "A/G,A/C/G,T", the combination of which is threonine (T), alanine (A ), asparagine (N), aspartic acid (D), serine (S) or glycine (G). The selection of amino acids encoded by degenerate codons is shown on the right and marked with an X.

Additionally and preferably _{, the random sequence cassette for the V H} -CDR3 sub-library may comprise nucleic acid sequences of different lengths. For example, _{the random sequence cassette for the V H} -CDR3 sub-library may be of any length from 10 to 30 amino acids, preferably from 10 to 25 amino acids, more preferably from 10 to 20 amino acids. Thus, as shown in Table 1, V _H -CDR3 sub-oligonucleotide for generating a random sequence cassette for library oligonucleotides D / GR / L and (G / A "NNK" between the sequence encoding the A / G /T/C, representing G/A/T/C, G/T) (eg, 4 to 10 repeats) (see also FIG. 3 ). The generation and diversity of light chain sequences is exemplarily presented in FIG. 4 .

[Table 1]

[Table 2]

Most typically, the oligonucleotides presented in Tables 1 and 2 are provided as single-stranded DNA, which is a backbone comprising a fixed sequence region (eg, 5'- (promoter-) for recombinant nucleic acids in the _{V L sub-library, etc.} 5' UTR - FW1 + CDR1 + FW2 + CDR2 + FW3) - (FW4)) can be converted to a double stranded DNA fragment using DNA polymerase I (Klenow fragment). It is also contemplated that the oligonucleotides presented in Tables 1 and 2 are also present with complementary oligonucleotides to form double stranded nucleic acids without the use of polymerases.

In some embodiments, the recombinant nucleic acid of the sub-library also comprises a nucleic acid sequence encoding a protein tag such that the peptide encoded by the recombinant nucleic acid can be isolated using a binding agent to the protein tag. For example, preferred protein tags include the FLAG tag (with the sequence motif DYKDDDDK), the Myc tag (with the sequence motif EQKLISEEDL) and the HA-tag. In some embodiments, protein tags can be repeated to enhance signal or increase detection (eg, 3 repeats of a FLAG tag (3X FLAG), etc.).

It is contemplated that some random sequence cassettes inserted into the recombinant nucleic acid of a sub-library may introduce frame shifts, nonsense mutations, and sequence(s) that destabilize the structure of the peptide encoded by the recombinant nucleic acid. Accordingly, in some embodiments, the inventors contemplate that the recombinant nucleic acids of a sub-library are tested in vitro so that any recombinant nucleic acids encoding labile or misfolded peptides can be removed from the library. For example, _{the recombinant nucleic acid of the V H} -CDR3 sub-library or V _L sub-library is a staphylococcus that independently binds a structured epitope of _{the V H} 3 domain or V _L (Vκ) domain of an immunoglobulin, respectively, independently of the CDR sequence. It can be tested for its binding affinity to protein A of Staphylococcus aureus or protein L of Finegoldia magna.

Any suitable method for screening a recombinant nucleic acid by binding affinity for protein A or protein L is contemplated. In one exemplary embodiment, the recombinant nucleic acid of the sub-library is transcribed into mRNA by in vitro transcription, and the 3'-end of the mRNA is coupled (covalently linked) to puromycin. Puromycin-coupled mRNA is translated in vitro such that a peptide transcribed from the puromycin-coupled mRNA is coupled to the mRNA via puromycin. Next, the peptide is contacted with protein A or protein L to identify peptides that effectively bind to protein A or protein L. Preferably, a peptide that binds to protein A or protein L with an affinity having _{a K D} ^{of 10 −6} M or less, preferably 10 ⁻⁷ M or less is selected and isolated. Once a peptide with high affinity for protein A or protein L is isolated, the cDNA of the isolated peptide can be generated via in vitro reverse transcription of mRNA coupled with puromycin and the peptide. The thus generated cDNA of the isolated peptide can then be inserted as a random sequence cassette to generate selected recombinant nucleic acids of the _{V H} -CDR3 sub-library or V _{L sub-library.} Alternatively, the recombinant nucleic acid of the sub-library may be in the form of mRNA, which is optionally pre-coupled with a puromycin molecule so that an in vitro transcription step for the recombinant nucleic acid (DNA format) may not be required are considered

Construction of scFv libraries from sub-libraries

The inventors also contemplate that at least two recombinant nucleic acids (members) of a sub-library may be recombined to form a recombinant scFv nucleic acid. In a preferred embodiment, each of the at least two recombinant nucleic acids (members) is selected from a different sub-library. For example, one recombinant nucleic acid may be selected from each _{of a V H} -CDR1/2 sub-library, a plurality of V _H -CDR3 sub-libraries and a V _{L sub-library.} For another example, one recombinant nucleic acid may be selected from each of two _{of a V H} -CDR1/2 sub-library, a plurality of V _H -CDR3 sub-libraries and a V _{L sub-library.} Preferably, at least one, more preferably all, of the recombinant nucleic acid(s) selected from the sub-library are preselected via affinity binding screening as described above.

Most typically, recombinant scFv nucleic acids can be constructed by recombination of portions of recombinant nucleic acids from sub-libraries. In such embodiments, the portion of the recombinant nucleic acid comprises a random sequence cassette inserted into the recombinant nucleic acid. Thus, for example, as a first step 1, V _H -CDR1 / 2 sub-part of a recombinant nucleic acid of the library is 5 '- [CDR1 + (FW2 ) + CDR2] -3' ( random sequence cassette is indicated by underlining) , preferably 5'-(part of FW1)-[ CDR1 + (FW2) + CDR2 ]-(part of FW3)-3', more preferably 5'- (promoter -5' UTR - FW1) + CDR1 + (FW2) + CDR2 + (part of FW3) -3' or 5'- (promoter -5' UTR - FW1) + CDR1 + (FW2) + CDR2 + (small linker) -3'. Similarly, for example, a portion of the recombinant nucleic acid of the _{V H} -CDR3 sub-library is 5′-[CDR3 ]-3′ (random sequence cassette is underlined), preferably 5′- (part of FW3) - CDR3 - (part of FW4)-3', more preferably 5'- (part of FW3) - CDR3 - (FW4)-3', or 5'- (small linker) - CDR3 - (FW4)-3 ' can be Portions of the recombinant nucleic acids from the V _H -CDR1/2 sub-library and the V _H -CDR3 sub-library are then isolated (eg, by PCR), and recombined (eg, using a restriction-ligation method). fused to, generated via recombinant PCR, etc.) _{to form a V H} domain recombinant nucleic acid. Thus, typically, the V _H domain recombinant nucleic acid will be in the structure of 5'-promoter - 5' UTR - FW1 + CDR1 + FW2 + CDR2 + FW3 - CDR3 -FW4 -3' (random sequence cassettes are underlined). . Optionally, the V _H domain recombinant nucleic acid may also comprise a nucleic acid sequence encoding a protein tag (eg, FLAG tag, Myc tag, HA tag, etc.) at its 3'-end as described above. In addition, the generated V _H domain, a recombinant nucleic acid can be placed in the V _H domain, a V _H domain library library member.

The V _H domain recombinant nucleic acid thus formed can be further recombine with the recombinant nucleic acid of the V _{L sub-library to form a recombinant scFv nucleic acid.} 5 presents one exemplary method of recombining sequences from a sub-library. As shown, also typically, _{a portion of the V H} domain recombinant nucleic acid and a portion of the recombinant nucleic acid of the V _L sub-library are fused into one recombinant scFv nucleic acid. For example, part of the V _H domain recombinant nucleic acid is 5'-promoter-[ 5'UTR-FW1 + CDR1 + FW2 + CDR2 + FW3-CDR3 -FW4 -3' (preferably a protein at its 3'-end) free of any nucleic acid encoding the tag, and _{the portion of the recombinant nucleic acid of the V L} sub-library is FW1' + CDR1 + FW2' + CDR2 + FW3' - CDR3 - FW4' (promoter and 5'- no UTR), so that the recombinant nucleic acid of the _{V L} sub-library can be fused to the 3′-end of a portion of the _{V H domain recombinant nucleic acid.} Thus, a typical recombinant scFv nucleic acid is a 5'-promoter - [5' UTR - FW1 + CDR1 + FW2 + CDR2 + FW3 - CDR3 -FW4]V _H - [FW1' + CDR1 + FW2' + CDR2 + FW3' - CDR3 - FW4']V _L -3'. It is highly preferred that the portion of the V _H domain recombinant nucleic acid and the portion of the recombinant nucleic acid of the V _L sub-library are placed in the same reading frame such that they encode a single polypeptide.

Preferably, _{the part of the V H} domain recombinant nucleic acid and the part _{of the recombinant nucleic acid of the VL} sub-library are fused via a nucleic acid encoding a linker (short peptide spacer fragment) between the two parts. Any suitable length and order of peptide sequences for linkers or spacers may be used. However, the length of the linker peptide is preferably 3 to 30 amino acids, preferably 5 to 20 amino acids, more preferably 5 to 15 amino acids. For example, we contemplate that glycine-rich sequences (eg, gly-gly-ser-gly-gly, etc.) are used to provide flexibility of the scFv between the _{V H} and V _{L domains.}

Optionally, the recombinant scFv nucleic acid may also comprise a nucleic acid sequence encoding a protein tag (eg, FLAG tag, Myc tag, HA tag, etc.) at its 3'-end as described above. In addition, the resulting recombinant scFv nucleic acid can be placed in an expression library as an expression library member.

In some embodiments, the recombinant scFv nucleic acids so formed are further screened based on their binding affinity, stability, pH sensitivity, and/or species cross-reactivity to one or more ligands of interest (eg, cancer antigens, neoepitopes, etc.) and/or ranked. For example, the stability of an scFv peptide encoded by a recombinant scFv nucleic acid can be analyzed by size exclusion chromatography, which measures the size of the peptide over time. For another example, the pH sensitivity and binding affinity of an scFv peptide encoded by a recombinant scFv nucleic acid can be assayed by contacting the scFv peptide with one or more ligands in different buffer conditions (pH, temperature, etc.).

For analysis and further isolation of a desired recombinant scFv nucleic acid from an expression library, the inventors contemplate that the recombinant scFv nucleic acid may be present in the form of an mRNA that is optionally pre-coupled with a puromycin molecule at the 3'-end of the mRNA. The puromycin-coupled mRNA can then be translated in vitro such that a peptide transcribed from the puromycin-coupled mRNA is coupled to the mRNA via puromycin. The peptide is then optionally contacted with one or more ligands in different buffer conditions (pH, temperature, etc.). Preferably, at pH 5.0 to 8.0, preferably at pH 6.0 to 8.0, more preferably at pH 6.5 to 8.0, the ligand has an affinity having _{a K D} ^{of 10 -6} M or less, preferably 10 ^-7 M or less. The peptides that bind are selected and isolated. Once the peptide with high affinity for the ligand(s) is isolated, the cDNA of the isolated peptide can be generated via in vitro reverse transcription of puromycin and mRNA coupled with the peptide.

In addition, the cDNA thus produced of the isolated peptide encoded by the recombinant scFv nucleic acid can be grafted onto and replaced with a portion of an immunoglobulin to form a recombinant immunoglobulin or fragment thereof. For example, the cDNA thus generated can be fused with the backbone of the immunoglobulin heavy chain constant region so that the variable regions of the heavy and light chains of the immunoglobulin can be replaced with scFvs formed by separate peptides. Alternatively, the present inventors also provide that the V _H portion (or V _H domain derived from recombinant nucleic acid) and V _L portion (or derived from recombinant scFv nucleic acid) of a recombinant scFv nucleic acid are grafted into and replaced with a portion of an immunoglobulin, It is contemplated that a recombinant immunoglobulin or fragment thereof can be formed. For example, a V _H portion (or V _H domain derived from a recombinant nucleic acid) and a V _L portion (or derived from a recombinant scFv nucleic acid) of a recombinant scFv nucleic acid are fused to a percent of an immunoglobulin heavy chain constant region or light chain constant region, respectively. to form immunoglobulins with variable regions specific for the desired ligand.

In these examples, the immunoglobulin can be of any type (eg, IgG, IgE, IgM, IgD, IgA and IgY) and of any class (eg, IgG1, IgG2, IgG3) to constitute different types of immunoglobulin. , IgG4, IgA1 and IgA2) are contemplated. Also, "antibody" may include, but is not limited to, human antibodies, humanized antibodies, chimeric antibodies, monoclonal antibodies, polyclonal antibodies. In this context, it should be appreciated that the systems and methods contemplated _{allow the generation of species-specific antibodies by grafting the isolated V H} and V _L domains into the remainder of the antibody of the desired species (eg, human). . In another example, the cDNA so generated can be fused with a nucleic acid encoding another portion of an immunoglobulin to form a fragment of an immunoglobulin. In this example, it is contemplated that the fragment of an immunoglobulin may be a Fab fragment, a Fab' fragment, F(ab')2, a disulfide linked Fv (sdFv) and an Fv. The inventors further contemplate that a portion of the cDNA so generated may be fused with a nucleic acid encoding another portion of an immunoglobulin to form any fragment comprising a _{V H} segment and/or a V _{L segment.}

In addition, the inventors contemplate that scFv moieties can also be used as targeting entities for a variety of proteins and non-protein molecules. For example, an scFv moiety can be coupled (typically coupled as a chimeric protein) to an ALT-803 type molecule to form a TxM entity with specific targeting ability (see, e.g., J Biol Chem . 2016 Nov 11;291(46):23869-23881). In another example, the scFv moiety can be coupled to a carrier protein (eg, albumin) that enables target-specific delivery of one or more drugs to a specific location in the tumor microenvironment where the drug is coupled to the carrier.

The present inventors construct sub-libraries through targeted diversification of random sequences and/or preselect members of sub-libraries, thereby minimizing diversity by eliminating sequences in which the expression library is unstable, does not bind, or misfolds. We further consider that at a sacrifice we can achieve a complexity of approximately 10 ^{12 .} Thus, while the approach described above for generating expression libraries provides a significant amount of sequence complexity, it is practical for screening binding agents/antibodies in small volumes. In addition, the approach described above for generating expression libraries has simplified the screening procedure for binding agents/antibodies. Traditionally, in vitro validation of any nucleic acid sequence (e.g., a randomized sequence) encoding a binding domain (or motif) has required the nucleic acid sequence converted to the _{F ab domain, thereby using the ligand of interest.} It could be tested via a pull-down assay. The methods presented herein allow ranking by affinity (e.g., Kd value), pH sensitivity, and species cross-reactivity (e.g., via surface plasmon resonance assays, etc.) without converting the _{nucleic acid sequences to F ab domains.} allows in vitro validation of nucleic acid sequences encoding binding domains (or motifs) via In addition, preselection of members from each library based on stability and sensitivity reduces the pools to be tested in the library so that the desired binder/scFv/antibody domain can be identified more quickly and efficiently. _{Thus, we also present high affinity binding agents (eg, with nanomolar and picomolar K d} ) from a high diversity pool using mRNA display techniques in which post-translational in vitro library members are screened for solid phase bound antigen. Consider methods for the separation of Once the binders have been identified, they can be further characterized by surface plasmon resonance spectroscopy with respect to _{affinity and K on} /K _{off characteristics, as further described below.} Viewed from a different perspective, the systems and methods contemplated allow for the rapid detection of binding agents and the generation of scFvs or antibodies in a process completely independent of the immune system in vivo.

Example

While any suitable diversification scheme may be contemplated to identify targeted diversification region(s) to maximize diversity while maintaining efficiency, the present inventors have determined that VH3/Vk1 has been used for randomization among the various domains of immunoglobulins. It could be one of the good candidate regions, and it was found that VH3 is considered a much more stable and soluble VH domain, and that Vk1 of the light chain is stable and soluble. Thus, the VH3/Vk1 randomized pair is considered to be more efficiently converted to full-size immunoglobulins. Therefore, we developed a preselection strategy using the VH3 and Vk1 frameworks. 1 presents one exemplary randomization strategy using VH3/Vk1 pairs. The protein sequences of at least 14 immunoglobulin molecules specific for one antigen are compared and analyzed. Among the 14 immunoglobulin molecules, the most stable and conserved sequence is used as a framework, and the loci of the variable sequence are analyzed for use as a randomized sequence and degree of randomization (eg, completely random, partially random, etc.).

The random on the basis of the screen strategy, the present inventors (see Fig. 2) CDR1 of the V _H domain, CDR2-n, CDR2-c, and (see Fig. 3) V _H CDR3 of the domains of various targeted for the screen sequence (randomization sequences, random oligos) were further generated. Methods may are described in the above, it is also shown in the schematic view of Figure 4, using a random oligonucleotide of V CDR1 of the _H domain, CDR2-n, CDR2-c, CDR3 and V _L CDR3 of the domain to produce a recombinant scFv nucleic acid . A high diversity library was constructed as exemplarily shown in FIG. 5 and discussed in more detail above.

Using a targeted diversification scheme as described in Figures 1-5 and a method for generating recombinant scFv nucleic acids, we generated a high diversity library, from which recombinant α-B7-H4 ₈₀₁ (α-B7-H4 , clone number 801) binder was isolated. The stability of recombinant α-B7-H4 ₈₀₁ was determined by analytical size exclusion chromatography over 15 minutes to assess any degradation or modification of the antibody. As shown in Figure 6 , the eluent of α-B7-H4 ₈₀₁ shows a single peak without any significantly smaller peaks, indicating that the α-B7-H4 ₈₀₁ binder produced by the method described above exhibits scFv or high stability. It shows that antibodies can be generated with

We have found that recombinant α-B7-H4 ₈₀₁ contains antibody components substantially similar to other commercially available α-B7-H4 antibodies (Rituxan®, LEAF®). Fragments of recombinant α-B7-H4 ₈₀₁ and two commercially available α-B7-H4 antibodies (Rituxan®, LEAF®) were analyzed via capillary electrophoresis sodium dodecyl sulfate (CE-SDS). As shown in Figure 7 , CE-SDS separations of recombinant α-B7-H4 ₈₀₁ antibody and two commercially available α-B7-H4 antibody (Rituxan®, LEAF®) fragments were obtained from light chain (middle peak) and glycolytic, respectively. It shows two prominent peaks corresponding to the sylated heavy chain (right peak). The left peak indicates the position of the 10 Kd standard marker for CE-SDS analysis.

The inventors have further discovered that various recombinant α-B7-H4 antibodies may exhibit different binding properties (eg, affinity, specificity, etc.) for target ligands. 8 _{shows two recombinant α-B7-H4 antibodies, α-B7-H4 801} and α-B7-H4 ₈₁₇ , tested for binding to B7-H4-expressing 293T cells as measured by mean fluorescence intensity (MFI). present. The results suggest that the α-B7-H4 ₈₀₁ antibody has a higher binding affinity to B7-H4-expressing 293T cells compared to the α-B7-H4 _{817 antibody, indicating that the differently randomized CDR domains bind to the ligand.} It shows that it can provide different binding affinities for The rightmost panel shows a control experiment with non-specific human IgG1 (hIgG1).

Recombinant α-B7-H4 antibodies were further tested to determine specific and effective binding to ligands (B7-H4) expressed on antigen presenting cells (APCs) using flow cytometry. As shown in Figure 9 , the recombinant α-B7-H4 antibody was able to specifically bind to the B7-H4 ligand (separating the peak from non-specific isotypic binding), indicating that the recombinant α-B7-H4 antibody is fully functional. indicates.

The inventors have also the scFv peptide (scFv B7-H4 ₈₀₁₎ and scFv B7-H4 ₈₀₁ and recombinant α-B7-H4 antibody _{(IgG α-B7-H4 801} ) produced by the same scFv peptides for B7-H4 surface A functional equivalent was found using a plasmon resonance assay. In this assay, Flag-tagged scFv B7-H4 ₈₀₁ is immobilized to the surface via α-Flag biotinylated antibody coupled with surface bound neutravidin. The surface immobilized scFv B7-H4 ₈₀₁ peptide is then contacted with an analyte comprising B7-H4. A similar assay was performed with the α-B7-H4 antibody. As shown in Figure 10 and Table 3 , scFv B7-H4 ₈₀₁ and IgG α-B7-H4 ₈₀₁ exhibit substantially similar affinity and binding characteristics to B7-H4, indicating that they are functionally equivalent. In addition, the binding affinity of in vitro translated peptides (scFvs) can be measured directly without grafting the peptides into the antibody backbone, so that more recombinant scFv nucleic acids in the expression library can be efficiently screened.

[Table 3]

Among a plurality of scFv peptides for B7-H4 with different random sequence cassettes in CDR1 to 3 of V _{H and CDR3 of V L} , we found that similarity in specific domains (specific random sequence cassettes) is similar to that of scFv peptides to ligands. It was tested whether it can be made to have similar binding characteristics for Five scFv peptides (801, 802, 905, 906 and 817) were tested for their binding affinity to B7-H4. Among them, as shown in Table 4, four scFv peptides (clones 801, 802, 905, 906) have similar CDR3 sequences. The _{four scFv peptides with similar random sequence cassettes in CDR3 of V H} show similar binding affinities to B7-H4 at both 25°C and 37°C (as shown in Table 5), which at least to B7-H4 In scFv peptides for this, _{the sequence within the CDR3 of V H} may be important for binding to ligand.

[Table 4]

[Table 5]

We also generated a plurality of scFv peptides (scFv IL-8) that bind to interleukin-8 (IL-8) using sub-libraries and expression libraries, and to IL-8 under different conditions (temperature and pH). affinity was tested. Exemplary scFv IL-8 peptides and their binding affinities measured at various conditions are presented in Table 6. Of the clones shown in Table 6, clones 49-7, 49-1 and 49-12 contain similar V _H CDR3 sequences, and clones 49-19, 49-37 and 49-25 contain similar V _H CDR3 sequences. . In addition, clones 49-3 and 43-2 contain similar V _H CDR3 sequences. In contrast to the scFv peptide to B7-H4, we found that the binding affinity of the scFv IL-8 peptide may not be critically dependent on the similarity of the random sequence in the CDR3 of _{V H .} For example, clones 49-18, 49-37 and 49-25 contain similar V _H CDR3 sequences, whereas the binding affinity of these sequences ( _{measured in K D} X 10 ^-9 M) is 0.894 X 10 ^It varies between -9 M and 25 X 10 ^{-9 M.}

[Table 6]

We further tested whether scFv IL-8 could effectively trap IL-8 and neutralize the effect of IL-8 by measuring neutrophil size. In general, neutrophils enlarge (eg, have a larger diameter, etc.) upon stimulation by IL-8 (as shown in FIG. 11 ). The present inventors found that the effect of IL-8 on neutrophil expansion was determined by either recombinant α-IL-8 antibody (mAb αIL-8 ₂₀₁ as shown in FIG. 12 , upper left graph) or several scFv IL-8 peptides (as shown in FIG. 12 ). It was found that most can be discarded upon addition of the same αIL-8 _#2 , αIL-8 _49-3 , αIL-8 _49-10 , bottom graph), indicating that the scFv IL-8 peptide is converted to free IL-8 in the medium. By binding, it is shown that the effect of IL-8 can be effectively neutralized.

IL-8 is a neutrophil chemotactic factor that allows neutrophils to migrate to the site of IL-8 release (eg, site of infection). To evaluate the functional effect of scFv IL-8 peptide, neutrophils were placed on the bottom of the insert with a porous membrane, and neutrophils attracted by IL-8 were able to trans-migrate from the insert to the medium through the porous membrane. It was placed in a medium containing various concentrations of IL-8. As shown in FIG. 13 , the number of migrated neutrophils increased by increasing the IL-8 concentration in the medium. Interestingly, the IL-8 effect was almost completely abolished upon addition of either the scFv IL-8 peptide (αIL-8 _43-2 ) or a recombinant IL-8 antibody derived from the scFv IL-8 peptide (mAb αIL-8 _{201 ).} .

14 depicts additional experimental data for various scFvs isolated using the mRNA display library as presented herein. More specifically, each data point represents an scFv for the target indicated at the bottom, and the affinity value for each scFv was determined. As can be readily observed, the (same) library generated a large number of high affinity binders for a variety of distinct targets, all of the binders were sub-microM, and many of the binders were in the sub-nanoM affinity range. . In addition, we also studied whether the affinity of scFvs could be preserved upon CDR grafting into human IgG. 15 depicts exemplary results for 29 CDR grafting experiments for selected scFvs grafted onto human IgG1 scaffolds. As can be observed from the results of FIG. 15 , the humanized IgG1 antibody maintained high specificity and affinity (typically within one size order).

Generation of Recombinant Entities Using Expression Libraries

A recombinant scFv nucleic acid or recombinant encoding one or more antibodies (eg, IgG, IgM, IgE, IgA, etc.) or fragment(s) thereof formed by recombination of the V _H domain recombinant nucleic acid and the recombinant nucleic acid of the V _{L sub-library.} It is further provided that the nucleic acid can be further inserted into an expression vector of a recombinant entity (eg, bacteria, yeast, virus) such that the recombinant scFv fragment can be produced by the recombinant entity or cells infected by the recombinant entity. are considered Any suitable recombinant entity capable of having and/or expressing a recombinant nucleic acid encoding a recombinant scFv fragment is contemplated. For example, a recombinant entity may comprise any suitable virus, including adenoviruses, adeno-associated viruses, alphaviruses, herpes viruses, lentiviruses, and the like. However, adenoviruses are particularly preferred. It is further preferred that the virus is a replication deficient and non-immunogenic virus typically achieved by targeted deletion of selected viral proteins (eg E1, E3 proteins). This desirable property can be further enhanced by deleting E2b gene function, and as recently reported high titers of recombinant virus can be achieved using genetically modified human 293 cells (see, e.g., J Virol . 1998 Feb; 72(2): 926-933]). Accordingly, the inventors contemplate that one desired viral vector may comprise a recombinant adenovirus genome with a deleted or non-functional E2b gene.

Alternatively, the recombinant entity may be a bacterium, and the expression vector is present at a level low enough to not elicit an endotoxic response in human cells and/or insufficient to induce CD-14 mediated sepsis when introduced into the human body. It may be a bacterial vector capable of being expressed in a genetically engineered bacterium that expresses an endotoxin. One exemplary bacterial strain with modified lipopolysaccharide includes ClearColi® BL21 (DE3) electrostatic cells. This bacterial strain is BLK with genotype F-ompT hsdSB (rB-mB-) gal dcm lon λ (DE3 [ lacI lac UV5-T7 gene 1 ind1 sam7 nin5 ]) msbA148 ΔgutQΔkdsD ΔlpxLΔlpxMApagPΔlpxPΔeptA . In this context, several specific deletion mutations (ΔgutQ ΔkdsD ΔlpxL ΔlpxMApagPΔlpxPΔeptA) _{encode the transformation of LPS to lipid IV A} , while one additional compensatory mutation (msbA148) allows cells to maintain viability in the presence of the LPS precursor lipid IVA. It should be recognized that there is These mutations result in deletion of the oligosaccharide chain from the LPS. More specifically, two of the six acyl chains are deleted. The six acyl chains of LPS are recognized by Toll-like receptor 4 (TLR4) complexed with myeloid differentiation factor 2 (MD-2), triggering the activation of NF-κB and the production of pro-inflammatory cytokines. . _{Lipid IV A, which} contains only four acyl chains, is not recognized by TLR4 and therefore does not trigger an endotoxic response. Although electrocompetent BL21 bacterium is provided as an example, the inventors contemplate that the genetically modified bacterium may also be a chemically competent bacterium. Alternatively or additionally, the recombinant entity is a yeast, and the expression vector is also in yeast, preferably Saccharomyces cerevisiae (eg GI-400 series recombinant immunotherapeutic yeast strains, etc.) It may be a yeast vector capable of being expressed.

The inventors have contemplated that a plurality of recombinant scFv nucleic acids and/or recombinant nucleic acids encoding one or more antibodies (eg, IgG, IgM, IgE, IgA, etc.) generate a series of recombinant entities (eg, recombinant viruses). It is contemplated that it may be used to increase the diversity of therapeutically effective recombinant entities. Preferably, a plurality of recombinant scFvs and/or nucleic acids encoding one or more antibodies (eg, IgG, IgM, IgE, IgA, etc.) (eg, binding kinetics, etc.), such that the top 30%, top 20%, top 10%, or 5% of scFv fragments or antibodies with the highest binding affinity or other binding characteristics are scFvs. It may be selected from a pool of fragments or antibodies. In some implementations, the selection process may include selection of high-pass fragments and enriching them through multiple rounds of selection. For example, in some embodiments, the top 30% of the scFv fragments or antibodies with the highest binding affinity can be selected in a first round of selection, and the top 30% of the scFv fragments with the highest binding affinity or The top 50% of antibodies (from round 1) can be selected in round 2 of selection, and so on. Although any suitable number of rounds of selection and percent pass-through (eg, top 30%, top 20%, etc.) can be used, the final set of scFv fragments or antibodies has the highest binding in the entire pool of scFv fragments or antibodies. It is preferred to be able to make up the top 30%, top 20%, top 10% or 5% of scFv fragments with affinity.

The obtained series of recombinant scFv nucleic acids and/or recombinant nucleic acids encoding one or more antibodies (eg, IgG, IgM, IgE, IgA, etc.) add a plurality of different scFv fragments and/or antibody binding to the same antigen. It can further be used to generate heterologous pools of recombinant entities (eg, recombinant viruses) that can be produced with Without wishing to be bound by any particular theory, we believe that this approach may increase the pool of high affinity candidate scFv fragments, thereby increasing the chances of identifying scFv fragments and/or antibodies capable of binding antigen most effectively. consider From a different point of view, recombinant scFv fragments or antibodies with the highest binding affinity may vary in vivo according to many variables (e.g., slight individual conformational variations of antigens between patients, different binding conditions (e.g., pH, other It may not be the most therapeutically effective scFv fragment or antibody because of environmental disorders, etc.), or because the recombinant scFv fragment or antibody with the highest binding affinity may have undesirable kinetic characteristics as a therapeutic antibody. Thus, by generating heterogeneous pools of recombinant entities using a series of nucleic acids encoding different scFv fragments or antibodies, there is an opportunity to identify therapeutically effective scFv fragments (even if not those with the highest affinity for antigen). . In addition, heterologous pools of antibodies that bind the same antigen (generated from heterogeneous pools of recombinant entities) have the effect of targeting antigens in vivo compared to homologous pools of antibodies that may all together lose effectiveness under certain in vivo conditions. can increase From a different point of view, the present inventors suggest that heterologous pools of recombinant entities, particularly recombinant viruses with recombinant nucleic acids encoding various scFv fragments or antibodies targeting the same antigen, are more therapeutically beneficial for treating patients with tumors and / or consider it to be effective.

In some embodiments, the expression vector may comprise a nucleic acid segment encoding a signaling peptide for extracellular secretion such that the resulting recombinant scFv or antibody can be secreted from the cell. While any suitable signaling peptide is contemplated, exemplary signaling peptides include a positively charged N-terminal region (n-region), a hydrophobic central region (h-region) and a neutral, polar C-terminal region (c-region). ), which may be cleaved during intracellular transport. The nucleic acid segment encoding the signaling peptide may preferably be located at the N-terminus of the recombinant nucleic acid encoding the scFv, optionally via a short linker (eg, a glycine-rich linker encoding nucleic acid).

Alternatively and/or additionally, the expression vector may further comprise one or more elements capable of inducing or boosting an immune response against the tumor and/or boosting the activity of the resulting scFv fragment. Thus, in some embodiments, the expression vector is a neoantigen(s), tumor-associated antigen(s) or tumor specific patient specific neoepitopes, costimulatory molecules, immune stimulating cytokines, recombinant immunoglobulin protein complexes and/or another nucleic acid segment encoding a checkpoint inhibitor. With respect to cytokines, any suitable cytokine capable of modulating an immune response (eg, increasing or decreasing T cell activity, etc.) is contemplated. Thus, contemplated costimulatory molecules are B7.1 (CD80), B7.2 (CD86), CD30L, CD40, CD40L, CD48, CD70, CD112, CD155, ICOS-L, 4-1BB, GITR-L, LIGHT, TIM3, TIM4, ICAM-1, LFA3 (CD58), and members of the SLAM family. In addition, the cytokine is IL-15 super potency coupled with at least one of IL-7, IL-15, IL-18, IL-21 and IL-22, or preferably both IL-7 and IL-21. a super agonist (IL-15N72D) and/or an IL-15 super agonist/IL-15Rα Sushi-Fc fusion complex such as ALT-803). Costimulatory molecules considered are B7.1 (CD80), B7.2 (CD86), CD30L, CD40, CD40L, CD48, CD70, CD112, CD155, ICOS-L, 4-1BB, GITR-L, LIGHT, TIM3, TIM4, ICAM-1, LFA3 (CD58), and members of the SLAM family. Exemplary checkpoint inhibitors include antibodies or binding molecules to CTLA-4 (particularly for CD8 ⁺ cells), PD-1 (particularly for CD4 ⁺ cells), the TIM1 receptor, 2B4 and CD160, e.g., ipil rimumab (ipilimumab) and nivolumab (nivolumab).

In some embodiments, recombinant entities can be used to generate scFv fragments and/or antibodies in vitro. For example, a recombinant bacterium or recombinant yeast can be induced and/or cultured to express an scFv fragment protein, wherein the expressed scFv fragment protein is purified for further use by any suitable method (eg, affinity binding purification, etc.). ) can be further purified and/or isolated. In other embodiments, the recombinant entity can be used to infect mammalian cells in vivo and/or ex vivo. For example, a recombinant virus having a recombinant nucleic acid encoding an scFv fragment can infect mammalian cells in vitro (or ex vivo) by co-incubating the recombinant virus with mammalian cells to produce scFv fragments in the mammalian cells. have. In this example, the mammalian cell can be any suitable mammalian cell capable of producing a protein from an exogenous nucleic acid that secretes a recombinant scFv fragment, and can be any stabilized cell line (human or non-human, e.g., HEK). -293 cells, CHO cells, etc.) or autologous cells obtained from a patient with a tumor (eg, autologous B cells isolated and/or expanded ex vivo, etc.).

Alternatively, a recombinant virus having a recombinant nucleic acid encoding the scFv fragment and/or antibody can be administered to a human or mammal (having a tumor) such that the scFv fragment and/or antibody can be produced and secreted in vivo. . In this embodiment, the recombinant virus may be formulated in any pharmaceutically acceptable carrier, preferably formulated as a sterile injectable composition having a viral titer of ^{10 4} to 10 ^{12 viral particles per dosage unit.} can However, alternative formulations are also considered suitable for use herein, and all known routes and modes of administration are contemplated herein. As used herein, the term “administering” a recombinant viral formulation refers to both direct and indirect administration of a recombinant viral formulation, wherein direct administration of the recombinant viral formulation is typically a health care professional (eg, a physician, nurse, etc.) Indirect administration includes providing or making available the recombinant viral formulation to a health care professional for direct administration (eg, via injection, infusion, oral delivery, topical delivery, etc.).

In some embodiments, the recombinant viral formulation is administered via systemic injection, including subcutaneous, subcutaneous injection, or intravenous injection. In other embodiments, where systemic injection may not be effective (eg, for brain tumors, etc.), it is contemplated that the recombinant viral formulation is administered via intratumoral injection.

With respect to the dose and schedule of administration of the recombinant viral formulation, the dose and/or schedule will depend on the tumor type, size, location, patient's health status (including, for example, age, sex, etc.), and any other relevant conditions. It is contemplated that this may vary. Although may vary, the dose and schedule can be selected and adjusted such that the recombinant virus does not provide any significant toxic effect on host normal cells but is sufficiently effective to induce the production of recombinant scFv fragments to treat tumors. For example, a contemplated dose of a recombinant viral formulation is at least 10 ⁶ viral particles/day, or at least 10 ⁸ viral particles/day, or at least 10 ¹⁰ viral particles/day, or at least 10 ¹¹ viral particles/day. am. In some embodiments, a single dose of the recombinant virus formulation is administered (administered at least once a day or twice a day for at least 1 day, at least 3 days, at least 1 week, at least 2 weeks, at least 1 month, or any other desired schedule. half dose) may be administered. In other embodiments, the dose of the recombinant viral formulation may be gradually increased over a period of time, or may be gradually decreased over a period of time. In another embodiment, several series of administrations of the recombinant viral formulation may be separated by an interval (e.g., once each for 3 consecutive days and once each for another 3 consecutive days with an interval of 7 days). administration, etc.).

It will be apparent to those skilled in the art that many more modifications than those already described are possible without departing from the inventive concept herein. Accordingly, the subject matter of the present invention is not limited except for the scope of the appended claims. Further, in interpreting both the specification and the claims, all terms are to be interpreted in the broadest possible manner consistent with the context. In particular, the terms "comprise" and "comprising" are to be construed in a non-exclusive manner to refer to an element, component or step, wherein the stated element, component or step is not explicitly recited. It indicates that it can be present, used, or combined with. As used throughout the description herein and in the claims that follow, the meaning of the singular term includes plural referents unless the context clearly dictates otherwise. Also, the meaning of “in” as used in the description herein includes “in” and “on” unless the context clearly dictates otherwise. Where a claim herein refers to at least one selected from the group consisting of A, B, C.... and N, the text is not A + N, or B + N, etc. should be construed as necessary.

Claims (48)

A recombinant virus comprising a recombinant nucleic acid comprising a member of an expression library encoding a separate antibody or antibody fragment, the recombinant virus comprising:
a member of the expression library,
create or provide (1) V _H -CDR1/2 sub-libraries, (2) a plurality of V _H -CDR3 sub-libraries, and (3) V _L sub-libraries (the sub-libraries (1) to ( 3) each comprises a plurality of members, each member of said sub-library comprising at least one random cassette having a plurality of degenerate base positions;
Recombining at least a portion of at least two members of the V _H -CDR1/2 sub-library, the plurality of V _H -CDR3 sub-libraries and the V _L sub-library to form an expression library member in the expression library,
Recombinant Virus. The recombinant virus of claim 1 , which is a genetically modified low immunogenicity virus. 3. The recombinant virus of claim 2, wherein the recombinant virus is human adenovirus serotype 5 having a mutation in at least one of genes E1A, E1B, E2B, E3. 4. The method of any one of claims 1 to 3, wherein the plurality of members of the _{V H} -CDR1/2 sub-library comprises a random cassette corresponding to at least one of a portion of a _{V H} CDR1 and a portion of a V _{H CDR2.} Recombinant Virus. 5. The method of any one of claims 1 to 4, wherein the plurality of members of the _{V H} -CDR1/2 sub-library comprises a plurality of random cassettes corresponding to _{at least a portion of the V H} CDR1 and a portion of the V _{H CDR2.} Recombinant Virus. 5. The recombinant virus of claim 4, wherein the plurality of members of the _{V H} -CDR1/2 sub-library comprises a plurality of random cassettes corresponding to at least a portion of the _{V H CDR2.} 7. The recombinant virus of any one of claims 1-6, wherein the plurality of members of the _{V H} -CDR3 sub-library comprises a random cassette corresponding to at least a portion of the _{V H CDR3.} Any one of claims 1 to 7, according to any one of items, V _H -CDR3 sub-recombinant virus which encodes the peptide is at least two random cassette of a member of the library having a different length. 9. The recombinant virus of claim 8, wherein the peptide has a length ranging from 10 to 20 amino acids. 10. The recombinant virus according to any one of claims 1 to 9, wherein a plurality of members of the sub-library have a consensus sequence. 11. The recombinant virus of any one of claims 1-10, wherein the plurality of members of the _VL sub-library comprises a random cassette in a portion of the _{VL CDR3.} 12. The method of any one of claims 1 to 11, wherein the recombination isolates at least a portion of a member of one of the _{V H} -CDR1/2 sub-library and the plurality of V _{H -CDR3 sub-libraries, and fuses them together} V _H domain in the library and form a V _H domain comprising the library member, the V _H domain of the library comprises a plurality of V _H domain library member, the recombinant virus. The method of claim 12, wherein the members of the expression library V _L sub-separates at least a portion of a member of the library, V _L sub-fusing one of the part of the members of the library and the V _H domain library members form an expression library members Recombinant virus produced by 14. The method according to any one of claims 1 to 13, wherein the recombination isolates at least a portion of a member of one of the _{V H} -CDR1/2 sub-library and the plurality of V _{H -CDR3 sub-libraries and fuses them together.} A recombinant virus comprising forming a first group of expression library members. 15. The recombinant virus of any one of claims 1-14, wherein the recombinant nucleic acid further comprises a nucleic acid fragment encoding a signaling peptide that promotes secretion of a separate antibody or antibody fragment. 16. The recombinant virus of any one of claims 1-15, wherein the separate antibodies or antibody fragments comprise scFvs. 18. The recombinant virus of claim 17, further comprising subcloning the expression library member to construct an IgG1 with scFv. 18. The recombinant virus of any one of claims 1-17, wherein the random cassette is generated using an oligonucleotide selected from SEQ ID NO:1 to SEQ ID NO:25. A method of generating a recombinant antibody comprising:
Creating or providing (1) a V _H -CDR1/2 sub-library, (2) a plurality of V _H -CDR3 sub-libraries, and (3) a V _L sub-library, the sub-library (1) to (3) each comprises a plurality of members; wherein each member of the sub-library comprises at least one random cassette having a plurality of degenerate base positions;
Recombining at least a portion of at least two members of the V _H -CDR1/2 sub-library, the plurality of V _H -CDR3 sub-libraries and the V _L sub-library to form an expression library member in the expression library, wherein the expression wherein the library comprises a plurality of expression library members, each expression library member encoding a separate antibody or antibody fragment; and
generating a recombinant viral vector comprising at least one expression library member;
Way. 20. The method of claim 19, wherein the recombinant viral vector is derived from a genetically modified low immunogenicity virus. The method of claim 20 , wherein the genetically modified low immunogenicity virus is human adenovirus serotype 5 having a mutation in at least one of genes E1A, E1B, E2B, E3. 22. The method of any one of claims 19-21, wherein the plurality of members of the _{V H} -CDR1/2 sub-library comprises a random cassette corresponding to at least one of a portion of a _{V H} CDR1 and a portion of a V _{H CDR2.} Way. 23. The method of any one of claims 19-22, wherein the plurality of members of the _{V H} -CDR1/2 sub-library comprises a plurality of random cassettes corresponding to _{at least a portion of the V H} CDR1 and a portion of the V _{H CDR2.} Way. 24. The method of claim 23, wherein the plurality of members of the _{V H} -CDR1/2 sub-library comprises a plurality of random cassettes corresponding to at least a portion of the _{V H CDR2.} 25. The method of any one of claims 19-24, wherein the plurality of members of the _{V H} -CDR3 sub-library comprises a random cassette corresponding to at least a portion of the _{V H CDR3.} 26. The method according to any one of claims 19 to 25, wherein _{at least two random cassettes of members of the V H} -CDR3 sub-library encode peptides of different lengths. 27. The method of claim 26, wherein the peptide has a length ranging from 10 to 20 amino acids. 28. The method of any one of claims 19-27, wherein the plurality of members of the _VL sub-library comprises a random cassette in a portion of the _{VL CDR3.} 29. The method of any one of claims 19-28, wherein the plurality of members of the sub-library have a consensus sequence. 30. The method of any one of claims 19-29, wherein each expression library member comprises a plurality of random cassettes. 31. The method of any one of claims 19-30, wherein the recombination isolates at least a portion of a member of one of the _{V H} -CDR1/2 sub-library and the plurality of V _{H -CDR3 sub-libraries, and fuses them together} comprises forming a V _H domain library member in the V _H domains and libraries, wherein the V _H domain library comprises a plurality of V _H domain library member. 32. The method of claim 31, V _L sub-step of separating at least a portion of a member of the library and the V _L sub-fusing one of the part of the members of the library and the V _H domain library members add to form the expression library members How to include. 33. The method of claim 32, wherein _{some of the members of the V L} sub-library are coupled via a sequence encoding a linker. 34. The method of claim 33, wherein the linker is a glycine-rich peptide. 35. The method according to any one of claims 19 to 34, wherein the recombination isolates at least a portion of a member of one of the _{V H} -CDR1/2 sub-library and the plurality of V _{H -CDR3 sub-libraries and fuses them together.} A method comprising forming a first group of expression library members. 36. The method of claim 35, further comprising a second group of expression library members, said second group comprising at least a portion of the members of the _{V L sub-library.} 37. The method of any one of claims 19-36, wherein the separate antibody or antibody fragment comprises an scFv. 38. The method of claim 37, further comprising subcloning the expression library member to construct an IgG1 with scFv. 37. The method of claim 36, further comprising subcloning each one member from each of the first and second groups to form an IgG1 having _{recombinant V H} and V _{L domains.} 40. The method of any one of claims 19-39, further comprising selecting a subset of expression library members based on at least one of affinity for the ligand, pH sensitivity and species cross-reactivity. 41. The method of claim 40, further comprising selecting a subset of expression library members that encode scFvs that bind ligand with a Kd of less than 50 nM. 42. The method of any one of claims 19-41, wherein the random cassette is generated using an oligonucleotide selected from SEQ ID NO:1 to SEQ ID NO:25. 43. The method according to any one of claims 19 to 42,
transcribing the expression library member into an mRNA fragment; and
The method further comprising coupling the puromycin molecule at the 3'-end of the mRNA fragment. 44. The method of any one of claims 19-43, further comprising contacting the recombinant virus with the recombinant viral vector with a mammalian cell. 45. The method of any one of claims 19-44, wherein contacting comprises administering the recombinant virus to the mammal. 46. The method of any one of claims 19-45, wherein the mammalian cells are autologous cells of a patient having a tumor, and contacting comprises co-incubating the autologous cells and the mammalian cells ex vivo. 19. Use of the composition of any one of claims 1 to 18 for generating a therapeutic recombinant antibody against a cancer neoepitope. 47. A method of isolating a high affinity binding agent having an affinity of 100 nM or less for an antigen, comprising contacting the antigen with a composition constructed by the method of any one of claims 19-46.

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