mRNA display antibody libraries and methods

Abstract

To provide methods for preparing high diversity nucleic acid libraries encoding antibodies and antibody fragments.SOLUTION: The high-diversity nucleic acid library comprises or is derived from (1) a VH-CDR1 / 2 sub-library, (2) a plurality of VH-CDR3 sub-libraries, and (3) a VL sub-library, each of which comprises a plurality of members. Preferably, each member of the sub-libraries comprises at least one random cassette that has a plurality of degenerate base positions. In an especially preferred embodiment, at least portions of at least two members of the VH-CDR1 / 2 sub-library, the plurality of VH-CDR3 sub-libraries, and the VL sub-library are recombined to form an expression library member in an expression library, where each member of the expression library encodes a distinct antibody or antibody fragment.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] This application is a subsidiary of the applicant's co-pending U.S. provisional patent application filed on November 20, 2017. Priority is claimed to Patent Application No. 62 / 588,914.

[0002] The field of the present invention is particularly related to mRNA display libraries and recombinant high affinity binding libraries. The use of mRNA display libraries to generate vectors has led to extremely high The present invention relates to compositions and methods for generating highly diverse antibody libraries. [Background technology]

[0003] The background description includes information that may be useful in understanding the present invention. Any information contained herein is prior art or relevant to the invention claimed herein. that any publication specifically or implicitly referenced is prior art; It is not something to be acknowledged.

[0004] All publications and patent applications herein are hereby incorporated by reference in their entirety. Any references incorporated by reference shall be deemed to have been incorporated by reference to the same extent as if they were specifically and individually indicated to be incorporated by reference. The definitions and usage of terms in the incorporated references are provided herein. If the definition of a term provided herein is inconsistent with or contradicts that provided herein, The definition applies, and definitions of the terms in the references do not apply.

[0005] Targeting tumor antigens or neoepitopes with high affinity, specific antibodies or binding molecules has been proven to be an effective method for treating cancer patients. Patient-specific and / or cancer-specific tumor antigens and / or neoepitopes are identified using omics data. Stable as identified through in vivo, in vitro, or in silico analysis Therefore, it is possible to select an antibody or binder that is highly likely to be soluble, functional, and applicable. There is an increasing demand for the creation of selectable antibody libraries or display libraries. Can high affinity specific antibodies or binding molecules be identified within the natural antibody pool? or may be derived from a natural antibody pool, but such identification or derived natural antibodies or binding sites are not intended to be limiting. The markers may develop such symptoms depending on the frequency or intensity of exposure to such antigens or neoepitopes. Such natural antibody diversity may be limited and therefore may not be as effective or specific. be.

[0006] One approach to solving these problems is recombinant phage display. Such an approach can be used to generate libraries with a reasonably high degree of diversity. This allows the preparation of libraries, but often requires multiple concentration steps for the binder. Moreover, despite the relatively large diversity of Binders tend to lack ideal affinity and stability. Moreover, diversity is usually The number of vectors is limited by practical considerations such as the amount of vector and transfection efficiency. Such approaches and other approaches are further described in, for example, WO 2006 / 0727 It can be optimized using multiple artificial selection pressures as described in Pamphlet No. 73. Such methods may improve stability characteristics, but may also result in significant loss of library - Operation and time are required.

[0007] In yet another approach, mRNA display can be performed, where the candidate The mRNA sequences encoding the binding molecules (typically scFvs) are purified at their 3' ends. The peptides encoded by the mRNA sequences bound to the cyclosporine molecules were then synthesized in vitro. Generated via translation, it directly binds mRNA to the protein encoded by the mRNA However, current mRNA display technology does not This advantageously avoids the problems associated with infection restrictions and allows for, at least conceptually, greater diversity. but may have poor structural integrity or stability, relatively low affinity, and / or cross-reactivity. The problem still remains: at least the selection of scFv from mRNA display To further refine the binding properties, VH-CDR3 spectratyping analysis was performed. Protein Engineering, Design & Selection (See 2015, vol. 28 no. 10, pp. 427-435.) However, such a process requires repeated analysis and may not be productive for all antigens. There may not be.

[0008] Therefore, mRNA display and other methods are used to generate candidate binders and Although methods are known to identify specific nucleotide sequences that have high structural integrity / stability, low affinity, and / or Highly diverse libraries with binders with low cross-reactivity remain elusive Therefore, the mRNA was used to rapidly generate stable recombinant high affinity binders. There remains a need for improved compositions, methods, and uses of A display libraries. exist. Summary of the Invention

[0009] The subject of the present invention is the development of stable and viable antibodies against various biomolecules, in particular cancer antigens or neoepitopes. Multiple antibody binding assays that allow for reliable and efficient identification of soluble, functional antibodies or binders. Various compositions, methods, and uses of high diversity nucleic acid libraries encoding antibodies or antibody fragments. Thus, one aspect of the subject matter is directed to the use of a high-throughput PCR product encoding multiple antibodies or antibody fragments. The present invention also includes a method for generating a diverse nucleic acid library, each of which contains a plurality of members. It has three sub-libraries: (1) V H -CDR1 / 2 sublibrary, (2) Number of V H -CDR3 sublibrary, and (3) V L Will sub-libraries be created? Each member of the three sub-libraries has a plurality of degenerate base positions. Contains at least one random cassette. At least two members of the three libraries At least a portion of the bar is recombined to form an expression library member in the expression library. A library is formed that has a plurality of expression library members. Each expression library member encodes a fragment. In a preferred embodiment, the expression library The members are transcribed into mRNA fragments which are subsequently linked to a puromycin molecule at the 3' end. are combined.

[0010] In another aspect of the present subject matter, the inventors provide a composition having a plurality of nucleic acid libraries. The plurality of nucleic acid libraries includes: (1) V H - CDR1 / 2 sublibrary, (2) Multiple Vs H -CDR3 sublibrary, and (3) V L Include sub-libraries Each of the sub-libraries (1) to (3) includes a plurality of members, Each member of the sequence includes at least one random cassette having a plurality of degenerate base positions. .

[0011] In yet another aspect of the present subject matter, the inventors have created a highly diverse nucleic acid library. The present invention contemplates the use of the above compositions for the manufacture of

[0012] In yet another aspect of the inventive subject matter, the inventors provide a method for the preparation of a nucleic acid library member The present invention contemplates a high diversity nucleic acid library composition having a high diversity nucleic acid library member. The present invention relates to a recombinant nucleic acid comprising a plurality of random cassettes, each of which has a plurality of degenerate base positions. The plurality of random cassettes includes: (1) V H - CDR1 / 2 sublibrary, (2) Multiple Vs H -CDR3 sublibrary, and (3) V L From the sub-library Derived from at least two members from either of the two libraries.

[0013] In yet another aspect of the subject matter of the present invention, the inventors provide therapeutic antibodies against cancer neoepitopes. The use of highly diverse nucleic acid libraries to generate recombinant antibodies is contemplated.

[0014] In yet another aspect of the inventive subject matter, the inventors contemplate a method for producing a recombinant antibody. In this method, three sub-libraries, each having multiple members, are created: (1) V H -CDR1 / 2 sublibrary, (2) multiple V H -CDR3 sublibrary, and (3) V L A sub-library is created or provided. Three sub-libraries are provided: Each member of the sequence includes at least one random cassette having a plurality of degenerate base positions. At least a portion of the members of at least two of the three libraries are recombined to produce forming an expression library member in an expression library, which comprises a plurality of expression libraries; Each expression library member encodes a distinct antibody or antibody fragment. The method then includes producing a recombinant antibody or fragment thereof using the expression library members. continue.

[0015] In yet another aspect of the inventive subject matter, the inventors provide a composition constructed according to the above method. By contacting the antigen with the substance, a high affinity antibody having an affinity of 100 nM or less for the antigen is obtained. The present invention contemplates a method for isolating the reactive binder.

[0016] In yet another aspect of the inventive subject matter, the inventors have determined that the 100% 15 ... Recombinant nucleic acid fragments generated using selected oligonucleotides are contemplated.

[0017] In yet another aspect of the inventive subject matter, the inventors have determined that the 100% 15 ... Synthetic nucleic acid mixtures having selected nucleic acid sequences are contemplated.

[0018] Various objects, features, aspects and advantages of the present subject matter are set forth in the following detailed description, taken in conjunction with the accompanying drawings, in which: This will become more apparent from the following detailed description of the embodiments. [Brief description of the drawings]

[0019] [Figure 1] One exemplary randomization strategy using the VH3 / Vk1 pair is shown. [Diagram 2] 1 shows exemplary positions for sequence randomization in heavy chain CDR1 and CDR2. [Diagram 3]1 shows an exemplary sequence randomization in heavy chain CDR3. [Figure 4] An exemplary sequence randomization in the light chain CDR3 is shown with the nucleic acid sequence on the left and amino acid selection on the right. [Diagram 5] 1 shows an exemplary generation of hybrid nucleic acid elements by isolating and combining random cassettes of multiple recombinant nucleic acid segments. [Figure 6] FIG. 1 shows the results of size exclusion chromatography showing a single peak indicating stable protein expression of αB7-H4801. [Figure 7] Capillary electrophoresis sodium dodecyl sulfate (CE-SDS) data showing similar molecular behavior of αB7-H4801 compared to commercial antibodies. [Figure 8] 1 shows a graph depicting binding of in vitro selected αB7-H4 antibodies to B7-H4. [Figure 9] Figure 1 shows a graph of the functional analysis of in vitro selected αB7-H4 and αPD-L1 binders. [Figure 10] 1 shows a graph depicting the binding affinity of αB7-H4 scFv and αB7-H4 IgG1. [Figure 11] IL-8 activity assay by measuring the change in neutrophil size and the results thereof. [Figure 12] 1 shows a bar graph illustrating the neutralizing effect of αIL-8 antibodies on IL-8 activity to increase neutrophil size. [Figure 13] 1 shows an IL-8 activity assay and the results shown in a bar graph demonstrating the neutralizing effect of αIL-8 antibodies on IL-8 antibody activity by inhibiting neutrophil migration. [Figure 14] 1 shows exemplary results using the mRNA display library compositions presented herein with selected antigen targets. [Figure 15] Binders were identified using the mRNA display library compositions presented herein, and an exemplary graph showing the affinity of selected binders configured as scFv versus IgG is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] The present inventors are currently investigating antibodies or The targeted diversity of selected domains of the fragments is used to generate a high diversity nucleic acid library. By constructing a recombinant antibody or a fragment thereof, a specific and effective recombinant antibody or a fragment thereof can be produced or synthesized. To achieve this goal, the present inventors have now discovered that antibodies One or more domains or subdomains of the binder may be preselected, and the preselected Random cassettes within the selected domain or subdomain are used to generate multiple nucleic acid sublanes. The present inventors further discovered that it is possible to create a sub-library. The bars were recombined to allow for high diversity among library members while still allowing for the identification of cancer antigens. or neoepitopes (preferably cancer-specific, patient-specific neoepitopes or neoantigens). It is stable, soluble, functional, and suitable for use in vivo. Construct a highly diverse nucleic acid library to increase the probability of identifying antibodies / binders that are applicable to I discovered that it is possible.

[0021] Indeed, as will be shown in more detail below, the libraries presented herein are Typically, binders are below 100 nM in single- or two-pass concentration. Typically, K is less than 10 nM. d At least one binder to any antigen having Further, the contemplated system and method allows for the isolation of at least 10 9 At least Also 10 10 , at least 10 11 , at least 10 12, at least 10 13 At least Also 10 14 , at least 10 15 , or at least 10 16 Different library members The scFv library has a high diversity of 3D structures compared to conventional library construction. within a shortened time frame, thus substantially increasing the rate of antibody discovery. should be understood.

[0022] As used herein, the term "tumor" refers to a tumor located at one or more anatomical locations within the human body. One or more cancer cells, cancer tissues, malignant tumor cells, or malignant tumor tissues that may be detected or found. The term "telecommunications" refers to, and is used interchangeably with, textiles.

[0023] As used herein, the term "bind" is inclusive of the terms "recognize" and / or "detect." The term "interaction between two molecules" refers to "interaction between two molecules that occurs in a single molecule" and can be used interchangeably with them. 0 -6 M or less or 10 -7 K below M D It has high affinity.

[0024] As used herein, the term "provide" or "providing" means to manufacture or produce. Any act of making, placing, making available, or preparing for use Refers to and includes:

[0025] Construction of nucleic acid sublibraries In general, the structural components of an antibody (heavy chain, light chain, constant domain, variable domain) are For example, the variable domain (V H ) and light chain (V L ) Hato Together they constitute the epitope-binding domain that confers specificity to the antibody.H and V L Each of are composed of three complementarity determinants with unique amino acid sequences based on their specificity for antigens. The V H and V L Encoding the CDRs of A recombinant nucleic acid library for generating or identifying antibodies by randomizing sequences It has previously been contemplated that a V H Reach BiV L Complete randomization of all CDRs of the While there is a possibility that all randomized V H and V L However, antibodies (e.g., I When recombined to form a recombinant protein (e.g., gG1), it can be expressed either soluble or stably. Since it is not possible to generate all combinations of random sequences, we create all the randomized combinations Furthermore, we found that this creates inefficiencies in screening. Covering the entire range is not feasible due to the large number of possible library members. There is none.

[0026] Therefore, we have H and V L The subdomains are divided into two categories: V of an antibody (or the gene encoding that antibody) H Or V L A common framework among The framework region as well as the stability of the final peptide product (e.g., scFv, IgG1, etc.) At least partially or completely labile without significantly affecting the quality and / or solubility of the It is contemplated that the V H The targeted diversification regions are CDR1, CDR2-n (the N-terminal side of CDR2), and CDR1-n (the N-terminal side of CDR2). R2-c (the C-terminal side of CDR2), and at least a part of CDR3. In one embodiment, V L The targeted diversification region comprises at least a portion of the CDR3.

[0027] Thus, in one exemplary and particularly preferred embodiment of the present subject matter, a nucleic acid library V H and / or V L One or more randomizations in one or more targeted diversification regions The method can be carried out by generating a recombinant nucleic acid containing a nucleic acid sequence cassette. In an embodiment, we provide randomized recombination sequences within a single sub-library. The amount of a single sublibrary is too large to handle for rapid or timely screening. Each sub-library is then assigned to a library, avoiding the need to assign a library to each library, which can be impractical or inefficient. Different targeted diversification regions are used to preserve the diversity within the randomized targeted diversification region. Three different subpopulations with different sets of random sequence cassettes within the diversified region were Furthermore, the conserved regions between the targeted diversified regions are the largest Selected or designed for stability and solubility.

[0028] In one embodiment, the sub-library is H - Includes a CDR1 / 2 sub-library. V H -CDR1 / 2 sublibrary is V H At least a portion of CDR1 and / or V H A plurality of recombinant nuclei containing one or more random sequence cassettes corresponding to a portion of CDR2 As used herein, V includes nucleic acids (e.g., recombinant DNA). H Part of CDR1 The corresponding random cassettes are located within the region of the recombinant nucleic acid. The present invention relates to a method for producing a CDR1 region of a natural antibody, the method comprising the steps of: H The domain has at least V is structurally or functionally similar H Must be present to code for part of the domain Yes. For example, V H The recombined nucleic acids in the CDR1 / 2 sublibrary are as follows: The structure is (randomized regions are underlined, fixed sequenced regions are in brackets (put into): [ka] As used herein, UTR refers to an untranslated region and FW refers to a framework region (e.g., For example, FW1 is a first framework region that may be different from the second framework region (FW2). In this structure, the random sequence cassette is a CDR1 or The insertion may be in the CDR2 or, preferably, in both the CDR1 and CDR2 regions. In some embodiments, two or more random sequence cassettes, preferably two random sequence cassettes, The cassette can be inserted in the region of CDR2: CDR2-n (the 5'-end of CDR2 CDR-c (for the 3'-end of CDR2).

[0029] The sublibraries also support multiple V H -CDR3 sub-libraries. H - Each of the CDR3 sublibraries is H One or more sequences corresponding to at least a portion of CDR3 The method includes the step of: providing a plurality of recombinant nucleic acids (eg, recombinant DNA) having the random sequence cassettes described above. V H - As with the CDR1 / 2 sublibrary, V H - In CDR1 / 2 sublibrary The recombinant nucleic acid may have the structure as follows (the randomized regions are underlined: Fixed sequenced regions are in brackets): [ka] Preferably, V H -CDR1 / 2 sublibrary and / or V H -CDR3 Sublive The fixed sequence of the recombinant nucleic acid of the library (e.g., promoter-5'UTR-FW1+CDR1 +FW2+CDR2+FW3, FW4) are the most expressible fixed sequences, and single-chain variable A fragment (scFv), a modified form of a scFv, a full length immunoglobulin, or a portion of an immunoglobulin. The present invention is directed to a number of different forms of antibodies, including natural antibodies (e.g., For example, the most common and / or conserved sequences among IgG1 against various antigens may be used. Therefore, in a preferred embodiment, V H -CDR1 / 2 Sublicense Recombinant nucleic acid of Braly and V H - the fixed sequence of the recombined nucleic acid of the CDR3 sub-library is At least 70%, preferably at least 80%, more preferably at least 90% %Identical (shared).

[0030] The sublibraries are also L It may contain sub-libraries. L The sub-libraries are , V L Contains one or more random sequence cassettes corresponding to at least a portion of CDR3 A plurality of recombinant nucleic acids (e.g., recombinant DNA). H -CDR1 / 2 sublibrary As with V H - the recombinant nucleic acid in the CDR1 / 2 sublibrary has the following structure: (The randomized regions are underlined and the fixed sequenced regions are in brackets.) Can be inserted): [ka] Preferably, V L The fixed sequences of the recombinant nucleic acids of the sub-library are V H -CDR1 / 2 sub Library or V H - at least 70 of the recombined nucleic acids of the CDR3 sublibrary %, preferably at least 80%, more preferably at least 90% identical (shared can be).

[0031] Any randomized sequence can be considered to create a random sequence cassette. However, the inventors have H CDR1, CDR2, CDR3 and V L Domain CDR3 A strategic random sequence cassette for the purpose of the synthesis of a binding peptide (e.g., scFv, etc.) and The aim is to provide a high degree of complexity and a large potential binding surface when expressed as a single protein. For example, V H -Strategic for CDR1, CDR2 of CDR1 / 2 sublibrary The number of random sequence cassettes is 3 or less, preferably 2 or less, or more preferably 1 or less per cassette. More preferably, one random sequence (three or less, two or less, or one or more per cassette) Alternatively, the cassette may be a semi-random sequence cassette having a sequence encoding random amino acids. The position of the random sequence in the random cassette varies according to the random amino acid in the cassette. In another example, V H -Strategic for CDR3 of CDR3 sublibrary The random sequence cassettes are 4 or more, preferably 5 or more per cassette, or More preferably, 6 or more random sequences (4 or more, preferably 5 per cassette) 1 or more, or more preferably 6 or more random amino acids) are present , or more randomized sequences. L Sub-libraries The strategic random sequences for the CDR3 of the present invention are preferably 4 or more per cassette. At least 5, or more preferably 6 or more random sequences (at least 4 per cassette) Preferably, the sequence encodes 5 or more random amino acids, more preferably 6 or more random amino acids. ) may include a more randomized sequence.

[0032] In certain preferred aspects of the present subject matter, we provide a preferred Preferred random sequence cassettes are shown in Table 1 (V H -CDR1 / 2 sublibraries and V H - CDR3 sublibrary), and Table 2 (V L Regarding the sub-library It is contemplated that the nucleic acid sequence may be generated using the oligonucleotides presented in Tables 1 and 2. Each oligonucleotide is represented by an IUPAC ambiguity code, For example, the CDRs include random sequences having a degenerate code (highlighted). One oligonucleotide for the random sequence cassette contains the random sequence "RVT" This represents "A / G, A / C / G, T", the combination of which is threonine (T) , alanine (A), asparagine (N), aspartic acid (D), serine (S) or glycine The choice of amino acids coded for by degenerate codons is The choices are shown to the right and marked with an X.

[0033] Further and preferably, V H -Random sequence cassette for CDR3 sublibrary The vector may contain nucleic acid sequences of different lengths. For example, H -For CDR3 sublibraries The random sequence cassette for this purpose is 10 to 30 amino acids, preferably 10 to 25 amino acids. The length of the amino acid sequence may be any number of amino acids, more preferably 10 to 20 amino acids. As shown in FIG. 1, V H -Random sequence cassette for CDR3 sublibrary The oligonucleotides used to generate the kit were sequences encoding D / GR / L and A / G. Various repeats of "NNK" (standing for G / A / T / C, G / A / T / C, G / T) between columns (e.g., 4 to 10 repeats) (see also FIG. 3). The diversity is exemplarily shown in FIG.

[0034] [Table 1]

[0035] [Table 2]

[0036] [Table 3]

[0037] Most typically, the oligonucleotides presented in Tables 1 and 2 are single-stranded DNA. which uses DNA polymerase I (Klenow fragment) to synthesize double-stranded DNA A fragment containing a fixed sequenced region (e.g., V L Sub-libraries etc. Regarding the recombinant nucleic acid, 5'-(promoter-5'UTR-FW1+CDR1+FW2+ In addition, the fragments may be inserted into the scaffolds containing the CDR2+FW3)-(FW4)). The oligonucleotides shown also form double-stranded nucleic acids without the use of a polymerase enzyme. It is also contemplated that the nucleic acid may be present with a complementary oligonucleotide to achieve the desired sequence.

[0038] In some embodiments, the recombinant nucleic acids of the sub-library are also The encoded peptide can be isolated using a binder to the protein tag. For example, preferred protein tags include nucleic acid sequences encoding protein tags. Examples of the tag include the FLAG tag (having the sequence motif DYKDDDDK), the Myc tag (having the sequence motif Chief EQKLISEEDL), and HA-tag. In some embodiments, the protein tag may be repeated to enhance the signal or increase detection. For example, three repeats of the FLAG tag (3X FLAG) can be used.

[0039] Some random sequence cassettes inserted into the recombinant nucleic acids of the sublibrary are Chromosome shifts, nonsense mutations, and mismatched structures of peptides encoded by recombinant nucleic acids It is contemplated that stabilizing sequences may be introduced. Thus, in some embodiments In this paper, we present a method for the detection of any recombinant polypeptide encoding an unstable or misfolded peptide. The recombinant nucleic acids of the sub-library are in vitro purified so that the nucleic acids can be removed from the library. For example, V H -CDR3 sublibrary or V L Sabra The recombinant nucleic acids of the library are each independently selected from the V domain of an immunoglobulin, independent of the CDR sequence. H 3D Main or V L Staphylococcus aureus binds to a structured epitope in the Vκ domain Staphylococcus aureus Protein A or Finego Those against protein L of Finegoldia magna The binding affinity of the antibody can be tested.

[0040] Screening of recombinant nucleic acids by their binding affinity for Protein A or Protein L Any suitable method for cleaning is contemplated. In one exemplary embodiment, the sabra The recombinant nucleic acid of the library is transcribed into mRNA by in vitro transcription, and the mRNA is 'The end is bound (covalently) to puromycin. Puromycin-bound mRNA The peptides transcribed from the puromycin-transfected ribozyme were bound to the mRNA via puromycin. The cyclosporine-conjugated mRNA was transcribed in vitro. The peptide was then ligated to protein A. or Protein L to produce a peptide that efficiently binds to Protein A or Protein L. Preferably, 10 -6 M or less, preferably 10 -7 K below M D have Peptides that bind to Protein A or Protein L with a desired affinity are selected and isolated. Peptides with high affinity for Protein A or Protein L are isolated. The isolated peptide cDNA was then subjected to puromycin and peptide-bound mRNA synthesis. The isolated peptides thus produced can then be prepared by in vitro reverse transcription of A. The cDNA is H -CDR3 sublibrary or V L The selected set of sub-libraries It can be inserted as a random sequence cassette to generate a recombinant nucleic acid. The recombinant nucleic acid of the present invention is an in vitro transcription step for recombinant nucleic acid (DNA format Optionally, a puromycin molecule may be pre-assembled so that a pre-assembled ELISA kit (in the It is also contemplated that the polypeptide may exist in the form of an mRNA linked thereto.

[0041] Construction of scFv libraries from sub-libraries The present inventors further comprise: It is contemplated that the above-mentioned nucleic acids can be recombined to form a recombinant scFv nucleic acid. In the method, each of the at least two recombined nucleic acids (members) is from a different sub-library. For example, one recombinant nucleic acid is selected from the V H -CDR1 / 2 sublibraries, multiple V H -CDR3 sublibraries, and V L can be selected from each of the sub-libraries In another embodiment, the recombinant nucleic acid is H -CDR1 / 2 sublibraries, multiple V H -CDR3 sublibraries, and V L Select from each of two of the sublibraries. Preferably, at least one of the recombinant nucleic acids selected from the sub-library is At least one, and more preferably all, of the antibodies may be subjected to affinity binding screening as described above. is preselected.

[0042] Most typically, the recombinant scFv nucleic acid is a recombinant nucleic acid derived from a sub-library. In this embodiment, a portion of the recombinant nucleic acid may be constructed by recombining Thus, for example, the first step and And V H - a portion of the recombined nucleic acids of the CDR1 / 2 sub-library [ka] (random sequence cassettes are underlined), preferably [ka] More preferably, [ka] Similarly, for example, V H - a portion of the recombinant nucleic acid of the CDR3 sublibrary teeth, [ka] (random sequence cassettes are underlined), preferably [ka] More preferably, [ka] Next, V H -CDR1 / 2 sublibraries and V H -CDR3 subra A portion of the recombinant nucleic acid from the library is isolated (eg, by PCR) and obtained (e.g., fused via restriction-ligation methods, via recombinant PCR) (e.g., produced), V HA domain-recombined nucleic acid can be formed. Thus, typically, V H D The main recombinant nucleic acid is [ka] (The random sequence cassette is underlined.) H Domestic The recombinant nucleic acid may also contain a protein tag (e.g., F) at its 3′ end as described above. In addition, the nucleic acid sequence may include a nucleic acid sequence encoding a tag such as a LAG tag, a Myc tag, or an HA tag. V H The domain recombinant nucleic acid is H As a domain library member TE V H The domains may be arranged in a library.

[0043] The V thus formed H The domain recombinant nucleic acid further comprises a V L Recombination of sub-libraries The sub-library can be recombined with a nucleic acid to form a recombinant scFv nucleic acid. One exemplary method for recombining sequences from a nucleotide sequence is shown in FIG. , V H Part of the domain recombinant nucleic acid and V L A portion of the recombinant nucleic acids in the sub-library The scFv nucleic acid is fused to the nucleic acid. L The recombinant nucleic acids of the sub-library are H Domestic V can be fused to the 3' end of a portion of the recombinant nucleic acid. H Part of a domain recombination nucleic acid teeth, [ka] (Preferably without any nucleic acid encoding a protein tag at its 3' end) ), and V LA portion of the recombinant nucleic acids of the sub-library includes [ka] (without the promoter and 5'-UTR). The recombinant scFv nucleic acid is [ka] The structure of V H Part of the domain recombinant nucleic acid and V L Recombinant nucleic acids of the sub-library It is highly preferred that some of the sequences be placed in the same reading frame so as to code for a single polypeptide. I wish.

[0044] Preferably, V H Part of the domain recombinant nucleic acid and V L Recombinant nucleic acids of the sub-library One portion contains a nucleic acid encoding a linker (a short peptide spacer segment) between the two portions. The peptide sequence for the linker or spacer may be of any suitable length and However, the length of the linker peptide should not exceed 3 to 30 amino acids. Preferably, the amino acid sequence is 5 to 20 amino acids, more preferably 5 to 15 amino acids. For example, the present inventors have found that glycine-rich sequences (e.g., gly-gly-s er-gly-gly, etc.) H and V L Allows mobility of scFv between domains It is intended that the information be used in the following ways:

[0045] Optionally, the recombinant scFv nucleic acid may also have a tandem at its 3' end as described above. A nucleic acid sequence encoding a protein tag (e.g., a FLAG tag, a Myc tag, an HA tag, etc.) In addition, the recombinant scFv nucleic acid thus produced may be used in an expression library. The nucleic acid sequence can be arranged in an expression library as a set of members.

[0046] In some embodiments, the recombinant scFv nucleic acid so formed further comprises one or more their binding affinity to the ligand of interest (e.g., cancer antigens, neoepitopes, etc.) Screening and / or screening based on activity, stability, pH sensitivity, and / or species cross-reactivity. For example, scFv peptides encoded by recombinant scFv nucleic acids Stability was assessed by size exclusion chromatography, which measures the size of peptides over time. For another example, the scFv peptide encoded by the recombinant scFv nucleic acid can be analyzed. The pH sensitivity and binding affinity of the scTide were measured under different buffer conditions (pH, temperature, etc.). The Fv peptide may be analyzed by contacting it with one or more ligands.

[0047] Analysis and further isolation of the desired recombinant scFv nucleic acids from the expression library. Regarding isolation, the inventors have found that the recombinant scFv nucleic acid may exist in the form of mRNA. and optionally pre-linked with a puromycin molecule at the 3' end of the mRNA. Next, the peptides transcribed from the puromycin-bound mRNA are The puromycin-bound mRNA was then bound to the mRNA by the inactivation of puromycin. The peptides can then be transcribed in vitro and optionally covalently linked to one or more ligands. The contact is carried out under the buffer conditions (pH, temperature, etc.) suitable for the above purpose. Preferably, the pH is 5.0 to 8.0. , preferably pH 6.0 to 8.0, more preferably pH 6.5 to 8.0, -6 M or less , preferably 10 -7 K below M D A peptide that binds to a ligand with an affinity having Peptides having high affinity for the ligand are selected and isolated. When the peptide was isolated, the cDNA of the peptide was bound to puromycin and the peptide. It may be produced via in vitro reverse transcription of RNA.

[0048] Further, the isolated peptides encoded by the recombinant scFv nucleic acids so produced The cDNA is then grafted onto and substituted for portions of immunoglobulin to produce recombinant immunoglobulins or For example, the cDNA so generated can be used to generate fragments of immunoglobulins. The variable regions of the high and low chains can be replaced with scFvs formed by isolated peptides. Alternatively, the present inventors have also found that the fusion domain can be fused to an immunoglobulin heavy chain constant region framework, such as V of recombinant scFv nucleic acid H Part (or V H Domain derived from recombinant nucleic acid) and V L portion (or derived from a recombinant scFv nucleic acid) to replace a portion of an immunoglobulin. It is contemplated that recombinant immunoglobulins or fragments thereof can be produced. For example, recombinant sc Fv nucleic acid V H Part (or V H Domain derived from recombinant nucleic acid) and V L Partial (or recombinant (derived from scFv nucleic acid) is an immunoglobulin heavy chain constant region or light chain constant region, to form an immunoglobulin with a variable region specific for a desired ligand. do.

[0049] In these instances, immunoglobulins are differentiated by heavy or Any type of constant domain (e.g., IgG, IgE, IgM, IgD, IgA, and Ig Y) and any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and In addition, the term "antibody" may include human antibodies, humanized antibodies, and the like. The antibodies may include, but are not limited to, chimeric antibodies, monoclonal antibodies, and polyclonal antibodies. In this context, the isolated V H and V L Species-specific antibodies by grafting domains onto the remainder of an antibody of the desired species (e.g., human). It should be noted that this allows the creation of a The cDNA is then fused to nucleic acids encoding other portions of the immunoglobulin to produce the immunoglobulin. In this example, the immunoglobulin fragment can be a Fab fragment, a Fab 'Fragments, F(ab')2, disulfide-linked Fv (sdFv), and Fv The present inventors further contemplate that a portion of the cDNA thus produced may be used as an immunoglobulin. fused to nucleic acids encoding other portions of the V H Segment and / or V L Segment It is contemplated that any fragment may be formed that contains any of the components.

[0050] Furthermore, the inventors have demonstrated that the scFv portion can be used to bind to a variety of protein and non-protein molecules. For example, the scFv portion can be used as a targeting entity for the (usually as a chimeric protein) to bind to ALT-803-type molecules to demonstrate specific targeting ability. A TxM entity having the following structure may be formed (see, for example, J Biol Chem. 2016 N ov 11;291(46):23869-23881). The scFv portion is then conjugated to a carrier protein (e.g., albumin) so that the drug binds to the carrier. The present invention provides a method for the targeted delivery of one or more drugs to specific locations in the tumor microenvironment when administered intracellularly. It is possible.

[0051] We further diversified the sub-libraries through targeted diversification of random sequences. By constructing and / or preselecting members of the sublibrary, The current library removes unstable, non-binding, or misfolded sequences. This allows for a reduction of approximately 10% while minimizing the sacrifice of diversity. 12 that we can achieve the complexity of It is therefore contemplated that the above approach to generating an expression library will Screening binders / antibodies in small quantities while providing a meaningful scale of complexity In addition, the above approach to generating an expression library is practical for This simplifies the screening procedure for inhibitors / antibodies. In vitro validation of any nucleic acid sequence (e.g., a randomized sequence) encoding The fusion of nucleic acid sequences ab domain, and then the binding affinity is The antibodies could then be tested via pull-down assays with a ligand of interest. The method shown comprises converting a nucleic acid sequence into F ab Without converting to a domain, affinity (e.g., Kd value), pH sensitivity, and species cross-reactivity (e.g., via surface plasmon resonance assays). Through ranking by (i.e., by In addition, each library based on stability and sensitivity is validated. Pre-selection of members from the library allows for faster identification of the desired binder / scFv / antibody domain. The pools to be tested in the library can be rapidly and efficiently identified. Therefore, we also determined that the library members after in vitro translation Using mRNA display technology, in which the vector is screened against a solid-phase-bound antigen, Select high affinity binders (e.g., nano- and picomolar K) from the diversity pool. d (having Once binders are identified, they can be used to detect, detect, and / or detect the presence of, or absence of, any of the binders described further below. As mentioned above, affinity and K on / K off The properties were investigated by surface plasmon resonance spectroscopy. In another aspect, the contemplated systems and methods can be further characterized as in vivo immunoassays. Rapid detection of binders and identification of scFv or antibody in a process completely independent of the immune system. Allows for the creation of the body. EXAMPLES

[0052] Working Example To maximize diversity while maintaining efficiency, the diversification region(s) to be targeted While any suitable diversification scheme for identifying VH3 / Vk1 is a good candidate region for randomization among various immunoglobulin domains. VH3 is considered to be the most stable and soluble VH domain, and the light chain We found that Vk1 of VH3 / Vk1 was stable and soluble. It is contemplated that the antibody will more efficiently convert the antibody to full-sized immunoglobulins. Therefore, we developed a pre-selection strategy using the VH3 and Vk1 frameworks. FIG. 1 shows one exemplary randomization strategy using a VH3 / Vk1 pair. The protein sequences of at least 14 immunoglobulin molecules specific for the antigen of The most stable and conserved sequence among 14 immunoglobulin molecules is the framework. The randomized sequences and randomization sequences are used as a benchmark to analyze the positions of the variable sequences. The degree of randomness (e.g., completely random, partially random, etc.) used.

[0053] Based on this randomization strategy, we further H CDR1, CD R2-n, CDR2-c (see Figure 2) and V H CDR3 of the domain (see Figure 3 (This refers to targeted diversification sequences (randomized sequences, random oligos) V H Domain CDR1, CDR2-n, CDR2-c, CDR3, and V L The process of producing a recombinant scFv nucleic acid using random oligos of the CDR3 of the domain is described above. and is also shown in the schematic diagram of FIG. 4. The high diversity library is shown in FIG. It was constructed as illustratively shown and discussed in more detail above.

[0054] Targeted diversification to generate recombinant scFv nucleic acids as described in Figures 1-5. Using the amplification scheme and method, the inventors have generated a high diversity library from which Recombinant α-B7-H4 801 (α-B7-H4, clone number 801) binder Recombinant α-B7-H4 was isolated. 801 The stability of the antibody was evaluated to assess the degradation or modification of the antibody. The results were determined by analytical size exclusion chromatography for 15 minutes. As described above, α-B7-H4 801 The elution of The peaks shown are those of α-B7-H4 produced by the above method. 801 Binder These results indicate that it was possible to generate scFv or antibodies with high stability.

[0055] The present inventors have developed recombinant α-B7-H4 801 However, other commercially available α-B7-H4 antibodies (Ritz It has been found that the antibody component is substantially similar to that of the IL-11 antibody (Xanthate (registered trademark), LEAF (registered trademark)). Recombinant α-B7-H4 801 and two commercially available α-B7-H4 antibodies (Rituxan ( Fragments of LEAF® and LEAF® were analyzed by capillary electrophoresis using sodium dodecyl sulfate. As shown in FIG. 7, the recombinant α- B7-H4 801 antibody and two commercially available α-B7-H4 antibodies (Rituxan®, L CE-SDS separation of the EAF® fragment showed two prominent peaks, each of which was a light The peaks on the left correspond to the glycosylated heavy chain (middle peak) and the glycosylated heavy chain (right peak). indicates the position of the 10 Kd standard marker for CE-SDS analysis.

[0056] The present inventors further demonstrated that various recombinant α-B7-H4 antibodies have different binding activities against the target ligand. It was found that the binding characteristics (e.g., affinity, specificity, etc.) of the antibody can be obtained by the method of the present invention. Binding to B7-H4 expressing 293T cells was tested, as measured by mass fluorescein intensity (MFI). The two recombinant α-B7-H4 antibodies tested are α-B7-H4 801 and α-B7-H 4 817 The results show that α-B7-H4 801 The antibody, α-B7-H4 817 Compared to antibodies It was shown that it has a higher binding affinity to B7-H4 expressing 293T cells compared to B7-H4 expressing 293T cells. The separately randomized CDR domains exhibit different binding affinities to ligands. The rightmost panel shows that non-specific human IgG1 (hIgG 1) shows a control experiment.

[0057] The recombinant α-B7-H4 antibody was further used to identify antigen-presenting cells (APCs) using flow cytometry. (APC) to determine specific and efficient binding to the ligand (B7-H4). As shown in FIG. 9, the recombinant α-B7-H4 antibody Can specifically bind B7-H4 ligand (peaks out from non-specific isotype binding) (isolated), indicating that the recombinant α-B7-H4 antibody is fully functional. There are.

[0058] The present inventors also investigated the effect of B7-H4 (scFv B7-H4 801 ) scFv peptide and scFv B7-H4 801 Recombinant scFv peptides produced α-B7-H4 antibody (IgG α-B7-H4 801 ) was measured by surface plasmon resonance assay. In this assay, the Flag tag was found to be functionally compatible with the Glycated scFv B7-H4 801 α-F is conjugated with surface-bound neutravidin The scFv is then immobilized on the surface via the lag biotinylated antibody. B7-H4 801The peptide is contacted with an analyte that contains B7-H4. was performed with α-B7-H4 antibody. As shown in FIG. 10 and Table 3, sc Fv B7-H4 801 and IgG α-B7-H4 801 has a substantial effect on B7-H4 These results suggest that the α- and β-terminal domains of the α-terminal β-terminal α ... Furthermore, the binding affinity of in vitro translated peptides (scFv) was evaluated by integrating the peptides into the antibody scaffold. The number of recombinations in the expression library can be measured directly without transplanting the The scFv nucleic acids can be efficiently screened.

[0059] [Table 4]

[0060] V H CDR1-3 and V L Various random sequence cassettes in CDR3 of Among multiple scFv peptides against B7-H4, the present inventors identified specific domains (particularly Similarity in the sequence of the scFv peptide (a random sequence cassette) determines the affinity of the scFv peptide for the ligand. We tested whether it is possible to make five scFv peptides (80 1, 802, 905, 906, and 817) and their binding affinity to B7-H4 Among them, four scFv peptides were tested, as shown in Table 4. The clones (clones 801, 802, 905, and 906) have similar CDR3 sequences. H These four scFv peptides have the same random sequence cassette in the CDR3 of The antibodies showed similar binding affinity for B7-H4 at both 25°C and 37°C (Table 5). As shown in Figure 1, this is at least in the scFv peptide against B7-H4. In V H that sequences in the CDR3 of may be important for binding to a ligand This shows that.

[0061] [Table 5]

[0062] [Table 6]

[0063] The present inventors also used the sub-libraries and the expression library to identify interleukin (IL) 1. By producing multiple scFv peptides that bind to IL-8 (interleukin-8) (scFv IL-8), The affinity for IL-8 was tested under various conditions (temperature and pH). The Fv IL-8 peptides and their binding affinities measured under various conditions are shown in Table 6. Among the clones shown in Table 6, clones 49-7, 49-1 and 49-12 are similar V H Contains CDR3 sequences, clones 49-19, 49-37 , and 49-25 are similar V H In addition, clone 49-3 and and 43-2 are similar V H Contains the CDR3 sequence. In contrast to the VFv peptide, we found that the binding affinity of the scFv IL-8 peptide was H of May not be critically dependent on similarity in random sequences in CDR3 For example, clones 49-18, 49-37, and 49-25 were found to have similar V H While containing CDR3 sequences, the binding affinity (K D X10 -9 M smell The unit of measurement is 0.894X10 -9 M and 25X10 -9 It varies between M.

[0064] [Table 7]

[0065] The present inventors further demonstrated that scFv IL-8 inhibits I We tested whether we could neutralize the effects of IL-8 by efficiently capturing IL-8. In general, neutrophils are expanded (e.g., larger linear cells) when stimulated by IL-8. diameter) (as shown in FIG. 11). Such IL-8 effects were confirmed by recombinant α-IL-8 antibody (mAb αIL-8 201 ,Figure 12 As shown in the graph in the upper left) or some scFv IL-8 peptides ( Alpha IL-8 #2 , αIL-8 49-3 , αIL-8 49-10 , as shown in FIG. As shown in the lower graph, the addition of s The cFv IL-8 peptide binds to free IL-8 in the medium, thereby promoting the release of IL-8. This indicates that the effect of the compound was effectively neutralized.

[0066] IL-8 induces neutrophils to migrate toward the site of IL-8 release (e.g., the site of infection). To evaluate the functional effects of scFv IL-8 peptides, Neutrophils were placed on the bottom of an insert with a porous membrane, and neutrophils attracted by IL-8 were Various concentrations of IL-8 were added to the inserts to allow transmigration through the porous membrane into the medium. As shown in FIG. 13, increasing the concentration of IL-8 in the medium Interestingly, such IL-8 The action of scFv IL-8 peptide (αIL-8 43-2 ) or scFv IL-8 peptide Recombinant IL-8 antibody (mAb αIL-8 201 ) added, almost Completely disappeared.

[0067] FIG. 14 shows the mRNA expression vectors isolated using the mRNA display library presented herein. Further experimental data for the various scFvs identified are presented. More specifically, each data point Each column represents the scFv against the target indicated at the bottom, and the affinity value for each scFv was determined. As can be seen, the (same) library allows multiple high-throughput assays against a variety of different targets. Binders with affinity were obtained, all of which were submicron and many of which were nanometers. In addition, the inventors also grafted CDRs onto human IgG. We also investigated whether the affinity of scFv can be maintained when the antibody is administered. 29 CDR grafting experiments on selected scFvs grafted onto a human IgG1 scaffold Exemplary results are shown. As can be seen from the results in Figure 15, the humanized IgG1 antibody has high specificity and retained affinity (usually within one order of magnitude).

[0068] Many variations besides those already described may be made without departing from the inventive concepts herein. It should be apparent to one skilled in the art that various configurations are possible. The present invention is not to be construed as being limited in any way except as set forth in the appended claims. In interpreting both the scope and scope of the In particular, the terms "comprise" and "comprising" should be interpreted in a non-exclusive manner to include elements, should be construed as referring to a component, step, or the like, and the referenced component, step, or or steps may be present or utilized, or other elements, configurations, or steps not expressly referenced The term "combined" refers to any element or step that may be combined with any other element or step as defined in this specification and the following claims. As used throughout the text, the meanings of "a," "an," and "the" shall be clear from the context. Unless otherwise indicated, the term "reference" includes plural references. As stated above, the meaning of "in" is "i" unless the context clearly dictates otherwise. n" and "on." When referring to at least one of the items selected from the above, the text may refer to A+N, B+N, etc. The term "group" should be construed as requiring only one member from the group, rather than, for example, any one element of the group.

Claims

1. A method for producing a recombinant antibody that binds to an antigen with high affinity, comprising the steps of: generating or providing (1) a VH-CDR1 / 2 sub-library, (2) a plurality of VH-CDR3 sub-libraries, and (3) a VL sub-library, each of said sub-libraries (1)-(3) comprising 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 said VH-CDR1 / 2 sub-library, said plurality of VH-CDR3 sub-libraries, and said VL sub-library to form expression library members in an expression library, said expression library comprising a plurality of expression library members, each expression library member encoding a distinct antibody or antibody fragment; Isolating expression library members encoding high affinity binding antibodies or antibody fragments using mRNA display; and generating an expression vector encoding said isolated expression library member; and producing said recombinant antibody from said expression vector.

2. The method of claim 1 , wherein the expression vector further comprises a nucleic acid fragment encoding a signal peptide that facilitates secretion of the distinct antibody or antibody fragment.

3. The method of claim 1, wherein the random cassette is generated using an oligonucleotide selected from the oligonucleotides shown in SEQ ID NO:1 to SEQ ID NO:

25.

4. 2. The method of claim 1, wherein a plurality of members of the VH-CDR1 / 2 sub-library comprises random cassettes corresponding to at least one of a portion of VH CDR1 and a portion of VH CDR2.

5. The method of claim 1, wherein a plurality of members of said VH-CDR3 sub-library comprises random cassettes corresponding to at least a portion of a VH CDR3.

6. The method of claim 1 , wherein a plurality of members of the VL sublibrary comprise random cassettes at a portion of a VL CDR3.

Images