mRNA display antibody library and method

Abstract

This invention provides improved compositions and methods for mRNA display libraries for the rapid production of stable recombinant high-affinity binders. [Solution] A composition, method, and use of recombinant viruses and / or recombinant viral vectors encoding distinct antibodies or antibody fragments prepared from a highly diverse nucleic acid library are disclosed. Preferably, the recombinant virus is a genetically modified low immunogenic virus, for example, an E2b deletion adenovirus. The highly diverse nucleic acid library is (1)V H -CDR1 / 2 sublibrary, (2) multiple V H -CDR3 sublibrary and (3)V L It includes or is derived from sublibraries, each of which includes multiple members. Preferably, each member of the sublibrary includes at least one random cassette having multiple degenerate base positions.

Description

[Technical Field]

[0001] The field of the present invention relates particularly to the use of mRNA display libraries for producing mRNA display libraries and recombinant high-affinity binders, and therefore to compositions and methods for highly diverse antibody libraries. [Background technology]

[0002] The background information includes information that may be useful in understanding the present invention. Not all information provided herein constitutes prior art to or related to the claimed invention, nor is any publication specifically or implicitly referenced herein considered prior art.

[0003] Any publications or patent applications described herein are incorporated by reference to the same extent as any individual publication or patent application is specifically and individually incorporated by reference. If the definitions and use of terms in an incorporated reference do not match or contradict the definitions provided herein, the definitions provided herein shall apply, and the definitions in the references shall not apply.

[0004] Targeting tumor antigens or neoepitopes with high-affinity, specific antibodies or binding molecules has proven to be an effective method for treating cancer patients. As an increasing number of patient-specific and / or cancer-specific tumor antigens and / or neoepitopes are identified in vivo, in vitro, or in silico via omics data analysis, there is a growing need to create antibody libraries or display libraries that can select stable, soluble, functional, and adaptable antibodies or binders with high probability. High-affinity, specific antibodies or binding molecules can be identified in or derived from the natural antibody pool; however, such identified or derived natural antibodies or binders may not be effective or specific because the diversity of such natural antibodies may be limited depending on the frequency or intensity of exposure to such antigens or neoepitopes.

[0005] One approach to address these problems is the use of recombinant phage display libraries. While this approach allows for the creation of libraries with moderately high diversity, it often requires multiple enrichments for the binder, making it time-consuming and labor-intensive. Furthermore, despite relatively high diversity, the binders tend to lack ideal affinity and stability. Additionally, diversity is typically limited by practical considerations such as library volume and transfection efficiency. Such approaches, and others, can be further optimized using multiple artificial selection pressures, as described, for example, in the international publication brochure 2006 / 072773. While such methods may improve stability characteristics, they require a significant amount of library manipulation and time.

[0006] In yet another approach, mRNA display can be performed. Here, mRNA sequences encoding candidate binding molecules (typically scFv) are bound to puromycin molecules at their 3' ends, and the peptide encoded by the mRNA sequence is generated via in vitro translation, producing a fusion product in which the mRNA is directly bound to the protein encoded by the mRNA. However, while current mRNA display techniques advantageously circumvent the problems associated with transfection limitations and allow for greater diversity, at least conceptually, problems with structural integrity or stability, relatively low affinity, and / or cross-reactivity still remain. To further improve at least selected binding properties of scFv from mRNA display, VH-CDR3 spectrtyping analysis has been performed (see Protein Engineering, Design & Selection, 2015, vol.28 no.10, pp.427-435). However, such processes require repeated analyses and may not be productive for all antigens.

[0007] Therefore, although methods for constructing and identifying candidate binders using mRNA display and other methods are known, libraries with high diversity from binders possessing high structural integrity / stability, low affinity, and / or low cross-reactivity remain unrealized. Thus, there is still a need for improved compositions, methods, and uses of mRNA display libraries for the rapid production of stable recombinant high-affinity binders. [Overview of the project] [Means for solving the problem]

[0008] The subject matter of the present invention relates to various compositions, methods, and uses of highly diverse nucleic acid libraries encoding multiple antibodies or antibody fragments that enable reliable and efficient identification of stable, soluble, and functional antibodies or binders against various biomolecules, particularly cancer antigens or neoepitopes. Accordingly, one aspect of the subject matter includes a method for preparing a highly diverse nucleic acid library encoding multiple antibodies or antibody fragments. In this method, three sublibraries, each having multiple members: (1)V H -CDR1 / 2 sublibrary, (2) multiple V H -CDR3 sublibrary and (3)V L Sublibraries are prepared or provided. Each member of the three sublibraries includes at least one random cassette having multiple degenerate base positions. At least a portion of at least two members of the three libraries are recombined to form expression library members in the expression library, which has multiple expression library members. Each expression library member encodes a separate antibody or antibody fragment. In a preferred embodiment, the expression library members are transcribed into mRNA fragments, which are then bound to a puromycin molecule at their 3' end.

[0009] In another aspect of the subject matter of the present invention, the inventors intend a composition having a plurality of nucleic acid libraries. The plurality of nucleic acid libraries are (1)V H -CDR1 / 2 sublibrary, (2) multiple V H -CDR3 sublibrary and (3)V L Includes sublibraries. Each of sublibraries (1) to (3) contains multiple members, and each member of a sublibrary contains at least one random cassette having multiple degenerate base positions.

[0010] In yet another aspect of the subject matter of the present invention, the inventors intend to use the above composition for producing a highly diverse nucleic acid library.

[0011] In yet another aspect of the subject matter of the present invention, the inventors contemplate a highly diverse nucleic acid library composition having a plurality of nucleic acid library members. The highly diverse nucleic acid library members each comprise a recombinant nucleic acid comprising a plurality of random cassettes having a plurality of degenerate base positions. The plurality of random cassettes are as follows: (1) V H -CDR1 / 2 sub-library, (2) a plurality of V H -CDR3 sub-library, and (3) at least two members from either of two libraries from the V L sub-library.

[0012] In yet another aspect of the subject matter of the present invention, the inventors contemplate the use of a highly diverse nucleic acid library for generating therapeutic recombinant antibodies against cancer neoepitopes.

[0013] In yet another aspect of the subject matter of the present invention, the inventors contemplate a method for generating 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-library, and (3) V L sub-library are generated or provided. Each member of the three sub-libraries comprises at least one random cassette having a plurality of degenerate base positions. At least a portion of at least two members of the three libraries are recombined to form expression library members in an expression library, which has a plurality of expression library members. Each expression library member encodes a distinct antibody or antibody fragment. Next, the method continues with generating a recombinant antibody or fragment thereof using the expression library members.

[0014] In yet another aspect of the subject matter of the present invention, the inventors contemplate a method for isolating a high-affinity binder having an affinity of 100 nM or less for an antigen by contacting an antigen with a composition constructed by the above method.

[0015] In yet another aspect of the subject matter of the present invention, the inventors intend to prepare recombinant nucleic acid fragments using oligonucleotides selected from Table 1 or Table 2 provided below.

[0016] In yet another aspect of the subject matter of the present invention, the inventors intend to provide a synthetic nucleic acid mixture having nucleic acid sequences selected from Table 1 or Table 2 provided below.

[0017] In yet another aspect of the subject matter of the present invention, the inventors intend a recombinant virus. This recombinant virus comprises recombinant nucleic acid comprising a member of an expression library encoding a distinct antibody or antibody fragment. The member of this expression library is (1)V H -CDR1 / 2 sublibrary, (2) multiple V H -CDR3 sublibrary and (3)V L A step of preparing or providing sublibraries, wherein each of these sublibraries (1) to (3) includes multiple members, and each member of these sublibraries includes at least one random cassette having multiple degenerate base positions, and V H -CDR1 / 2 sublibrary, multiple V H -CDR3 sublibrary, and V L It is prepared by a process in which at least a portion of at least two members of a sublibrary are rearranged to form expression library members in the expression library.

[0018] Typically, this recombinant virus is a genetically modified, low-immunogenic virus, which is most preferably human adenovirus serotype 5 having a mutation in at least one of the following genes: E1A, E1B, E2B, E3.

[0019] In some embodiments, V H -Multiple members of the CDR1 / 2 sublibrary are V H Part of CDR1 and V HIncludes a random cassette corresponding to at least one of the parts of CDR2. In such embodiments, V H -Multiple members of the CDR1 / 2 sublibrary are V H Includes multiple random cassettes corresponding to at least a portion of CDR2. In other embodiments, V H -Multiple members of the CDR1 / 2 sublibrary are V H At least a portion of CDR1 and V H Includes multiple random cassettes that correspond to part of CDR2.

[0020] In some embodiments, V H -Multiple members of the CDR3 sublibrary are V H Includes a random cassette equivalent to at least a portion of CDR3. H -At least two random cassettes of members of the CDR3 sublibrary are intended to encode peptides of varying lengths. Or and / or in addition, V L Multiple members of the sublibrary are V L Some CDR3s include random cassettes.

[0021] Typically, the rearrangement process is V H -At least some of the members of the CDR1 / 2 sublibraries and several V H -Isolate one of the CDR3 sublibraries and fuse them together, V H V in the domain library H The process of forming a domain library member, this V H Domain libraries are multiple V H The process includes forming a domain library member. In such an embodiment, the expression library member is V L Isolate at least some of the members of the sublibrary, V L Some of the members of the sublibrary, and V HIt is intended to be produced by fusing one of the domain library members to form an expression library member. In other embodiments, the recombination step is V H -At least some of the members of the CDR1 / 2 sublibraries and several V H -Includes the step of isolating one of the CDR3 sublibraries and fusing them together to form a first group of expression library members.

[0022] Optionally, recombinant nucleic acids may further contain nucleic acid fragments encoding signaling peptides that promote the secretion of distinct antibodies or antibody fragments.

[0023] In yet another aspect of the subject matter of the present invention, the inventors intend a method for producing recombinant antibodies. In this method, (1)V H -CDR1 / 2 sublibrary, (2) multiple V H -CDR3 sublibrary and (3)V L A sublibrary (each of these sublibraries (1) to (3) contains multiple members) is prepared or provided. Most preferably, each member of the sublibrary contains at least one random cassette having multiple degenerate base positions. Then, in this method, V H -CDR1 / 2 sublibrary, multiple V H -CDR3 sublibrary, and V L A recombinant viral vector can then be prepared, comprising the step of recombining at least a portion of at least two members of a sublibrary to form an expression library member in an expression library, wherein the expression library comprises a plurality of expression library members, each expression library member encoding a fraction of an antibody or antibody fragment.

[0024] Typically, this recombinant virus is a genetically modified, low-immunogenic virus, which is most preferably human adenovirus serotype 5 having a mutation in at least one of the following genes: E1A, E1B, E2B, E3.

[0025] In some embodiments, the random cassette is prepared using oligonucleotides selected from SEQ ID NOs: 1 to 25. Optionally, the recombinant viral vector further comprises nucleic acid fragments encoding signaling peptides that promote the secretion of distinct antibodies or antibody fragments.

[0026] Preferably, this method may further include the step of contacting a recombinant virus having a recombinant viral vector with mammalian cells. In some embodiments, the contact step includes administering the recombinant virus to a mammal. In other embodiments, the mammalian cells are autologous cells of a patient with a tumor, and the contact step includes co-incubating the autologous cells and mammalian cells ex vivo.

[0027] Various objects, features, aspects and advantages of the subject matter of the present invention will become more apparent from the following detailed description of preferred embodiments, along with the accompanying drawings. [Brief explanation of the drawing]

[0028] [Figure 1] We present one exemplary randomization strategy using VH3 / Vk1 pairs. [Figure 2] Exemplary positions for sequence randomization in heavy chain CDR1 and CDR2 are shown. [Figure 3] This shows exemplary sequence randomization in heavy chain CDR3. [Figure 4] The left image shows the nucleic acid sequence, and the right image shows an exemplary sequence randomization in light chain CDR3 with amino acid selection. [Figure 5] This example demonstrates the fabrication of a hybrid nucleic acid element by isolating and combining random cassettes of multiple recombinant nucleic acid segments. [Figure 6] The results of size exclusion chromatography showing a single peak indicating stable protein expression of αB7-H4801 are shown. [Figure 7] Capillary electrophoresis data of sodium dodecyl sulfate (CE-SDS) showing similar molecular behavior of αB7-H4801 compared to commercially available antibodies are presented. [Figure 8] This graph shows the binding of αB7-H4 antibodies selected in vitro to B7-H4. [Figure 9] The graphs show the functional analysis of αB7-H4 and αPD-L1 binders selected in vitro. [Figure 10] The graph shows the binding affinity of αB7-H4 scFv and αB7-H4 IgG1. [Figure 11] This paper presents an IL-8 activity assay using neutrophil size changes and its results. [Figure 12] The bar graph shows the neutralizing effect of αIL-8 antibodies on IL-8 activity, which increases neutrophil size. [Figure 13] The bar graph shows the neutralizing effect of αIL-8 antibody on IL-8 antibody activity by inhibiting neutrophil migration, along with the results of the IL-8 activity assay. [Figure 14] Exemplary results are shown using the mRNA display library compositions presented herein with respect to selected antigen targets. [Figure 15] The graph below illustrates the affinity of selected binders, configured as scFv versus IgG, identified using the mRNA display library compositions presented herein. [Modes for carrying out the invention]

[0029] The inventors have now discovered that specific and effective recombinant antibodies or fragments can be produced or identified by constructing a highly diverse nucleic acid library using targeted diversity in selected domains of antibodies or fragments encoded by members of the highly diverse nucleic acid library. To achieve this objective, the inventors have now discovered that one or more domains or subdomains of an antibody / binder can be pre-selected, and multiple nucleic acid sublibraries can be produced using a random cassette within the pre-selected domains or subdomains. The inventors have further discovered that by recombining members of the sublibraries, it is possible to construct a highly diverse nucleic acid library that allows for high diversity among library members while increasing the probability of identifying antibodies / binders that are stable, soluble, functional, and adaptable when used in vivo against cancer antigens or neoepitopes (preferably cancer-specific, patient-specific neoepitopes or neoantigens).

[0030] In fact, as will be shown in more detail below, the libraries presented herein typically have a binder of 100 nM or less, more typically 10 nM or less, in single-pass or two-pass enrichment. d This enables the isolation of at least one binder for any antigen having [specific characteristic]. Furthermore, the intended system and method shall be 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 15 , or at least 10 16 This enables the creation of scFv libraries with diverse library members within a significantly shorter timeframe compared to conventional library construction. Therefore, it should be understood that the speed of antibody discovery will substantially increase.

[0031] As used herein, the term “tumor” means one or more cancer cells, cancerous tissue, malignant tumor cells, or malignant tumor tissue that may be located in or found in one or more anatomical locations within the human body, and is used interchangeably with these terms.

[0032] As used herein, the term “binding” means the term “recognizing” and / or “detecting,” and can be used interchangeably with them, and the interaction between two molecules is 10 -6 M or less or 10 -7 K below M D It possesses high affinity.

[0033] As used herein, the terms “to provide” or “to offer” mean and include any act of manufacturing, producing, arranging, making available, or preparing for use.

[0034] Construction of nucleic acid sublibraries Generally, the structural components of an antibody (heavy chain, light chain, constant domain, variable domain) are closely related to their function. For example, the variable domain (V) in the heavy chain... H ) and light chain (V L Both of these constitute the epitope-binding domain that gives the antibody specificity. H and V L Each of these contains three complementarity-determining regions (CDRs, CDR1-3) that have unique amino acid sequences based on their specificity to the antigen. Therefore, V H and V L It had previously been considered that recombinant nucleic acid libraries for antibody production or identification could be prepared by randomizing the sequences encoding the CDRs of V. However, the inventors have found that V H and V L Completely randomizing all CDRs could introduce significant diversity into the library, while randomizing all V H and V LHowever, we found that generating all possible combinations of random sequences and screening all randomized combinations is inefficient because they are not necessarily soluble or stably expressed when recombined to form antibodies (e.g., IgG1). Furthermore, covering the entire diversity space is not practical due to the extremely large number of possible library members.

[0035] Therefore, the inventors of the present invention, V H and V L The subdomains of V fall into two categories: different antibodies (or the genes encoding those antibodies). H or V L The intention is to divide the region into a framework region that is generally common among them, as well as a targeted diversification region that can be at least partially or completely randomized without significantly affecting the stability and / or solubility of the final peptide product (e.g., scFv, IgG1, etc.). Preferably, V H The targeted diversification region includes at least a portion of CDR1, CDR2-n (the N-terminal side of CDR2), CDR2-c (the C-terminal side of CDR2), and CDR3. In a more preferred embodiment, V L The targeted diversified areas include at least a portion of CDR3.

[0036] Therefore, in one exemplary and particularly preferred embodiment of the subject matter of the present invention, the nucleic acid library is V H and / or V LThis can be produced by generating recombinant nucleic acids containing one or more random sequence cassettes in one or more targeted diversification regions. In one preferred embodiment, the inventors envision three different sublibraries, each having a different set of random sequence cassettes in different targeted diversification regions, so as to avoid a situation where there are too many randomized recombinant sequences in a single sublibrary, making it impractical or inefficient to handle the volume of a single sublibrary for rapid or timely screening. Furthermore, conserved regions between targeted diversification regions are selected or designed for maximum stability and solubility.

[0037] In one embodiment, the sublibrary is V H -Includes CDR1 / 2 sublibraries V H -CDR1 / 2 sublibrary is V H At least a portion of CDR1 and / or V H It comprises multiple recombinant nucleic acids (e.g., recombinant DNA) having one or more random sequence cassettes corresponding to a portion of CDR2. When used herein, V H The random cassette corresponding to a portion of CDR1 means that the random cassette is located within a region of recombinant nucleic acid, and within that region, the sequence encoding the CDR1 portion is the V of the natural antibody. H V that is at least structurally or functionally similar to the domain H It should exist to encode a part of the domain. For example, V H -The recombinant nucleic acids in the CDR1 / 2 sublibraries have the following structure (randomized regions are underlined, and fixed, sequenced regions are enclosed in parentheses): [ka] As used herein, UTR refers to the untranslated region and FW refers to the framework region (e.g., FW1 is the first framework region that may be different from the second framework region (FW2)). In this structure, the random sequence cassette can be inserted in the region of CDR1 or CDR2, or preferably, in both regions of CDR1 and CDR2. In some embodiments, two or more random sequence cassettes, preferably two random sequence cassettes, can be inserted in the region of CDR2: CDR2-n (with respect to the 5'-end side of CDR2) and CDR-c (with respect to the 3'-end side of CDR2).

[0038] The library may also include a plurality of V H -CDR3 libraries. Each of the V H -CDR3 libraries contains a plurality of recombinant nucleic acids (e.g., recombinant DNA) having one or more random sequence cassettes corresponding to at least a part of V H CDR3. V H -Similar to the V H -CDR1 / 2 library, the recombinant nucleic acids in the V

Chemical formula

[0039] The sublibrary may also contain V L sub-library. V L The sublibrary is V L It contains a plurality of recombinant nucleic acids (e.g., recombinant DNA) having one or more random sequence cassettes corresponding to at least a part of CDR3. V H - Similar to the CDR1 / 2 sublibrary, V H - The recombinant nucleic acids in the CDR1 / 2 sublibrary may have the following structure (the randomized regions are underlined and the fixed sequenced regions are enclosed in parentheses):

Chemical formula

[0040] Any randomized sequence can be considered for making a random sequence cassette, but the inventors have found that V H for CDR1, CDR2, CDR3 of and V L The strategized random sequence cassette for CDR3 of the domain is intended to provide high complexity and a large potential binding surface when expressed as a binding peptide (e.g., scFv, etc.). For example, V H-The strategically designed random sequence cassettes for CDR1 and CDR2 of the CDR1 / 2 sublibraries may be semi-random sequence cassettes having 3 or fewer, preferably 2 or fewer, or more preferably 1 random sequence (encoding 3 or fewer, 2 or fewer, or 1 random amino acid per cassette). The position of the random sequence in the random cassette may vary depending on the random amino acid in the cassette. In another example, V H -The strategized random sequence cassettes for CDR3 in the CDR3 sublibrary may contain more randomized sequences such that there are four or more, preferably five or more, or more than six random sequences per cassette (encoding four or more, preferably five or more, or more than six random amino acids per cassette). In yet another example, V L The strategized random sequences for the CDR3 sublibrary may include more randomized sequences such that there are four or more, preferably five or more, or more than six random sequences per cassette (encoding four or more, preferably five or more, or more than six random amino acids per cassette).

[0041] In a particular preferred embodiment of the subject matter of the present invention, the inventors have identified a preferred random sequence cassette for a sublibrary as shown in Table 1(V H -CDR1 / 2 sublibrary and V H -Regarding the CDR3 sublibrary), and Table 2 (V LIt is intended that these can be prepared using oligonucleotides presented in the sublibraries. As shown in Tables 1 and 2, each oligonucleotide contains a (highlighted) random sequence having a degenerate code, indicated as the IUPAC ambiguity code. For example, one oligonucleotide for the CDR1 random sequence cassette contains the random sequence "RVT," which represents "A / G, A / C / G, T," and its combination can encode one of threonine (T), alanine (A), asparagine (N), aspartic acid (D), serine (S), or glycine (G). The selection of amino acids encoded by the degenerate codon is shown on the right and indicated with X.

[0042] More preferably, V H - Random sequence cassettes for CDR3 sublibraries may contain nucleic acid sequences of different lengths. For example, V H -The random sequence cassette for the CDR3 sublibrary may be of any length of 10 to 30 amino acids, preferably 10 to 25 amino acids, more preferably 10 to 20 amino acids. Thus, as shown in Table 1, V H Oligonucleotides for constructing random sequence cassettes for the CDR3 sublibrary may contain various repeats of "NNK" (representing G / A / T / C, G / A / T / C, G / T) between the sequences encoding D / GR / L and A / G (e.g., 4 to 10 repeats) (see also Figure 3). The construction and diversity of light chain sequences are illustrated in Figure 4.

[0043] [Table 1]

[0044] [Table 2]

[0045] [Table 3]

[0046] Most typically, the oligonucleotides presented in Tables 1 and 2 are provided in single-stranded DNA, which is converted to double-stranded DNA fragments using DNA polymerase I (Kreno fragment) to a fixed sequenced region (e.g., V L Regarding recombinant nucleic acids such as sublibraries, they can be inserted into a backbone containing 5'-(promoter-5'UTR-FW1+CDR1+FW2+CDR2+FW3)-(FW4)). Furthermore, the oligonucleotides presented in Tables 1 and 2 are also intended to be present with complementary oligonucleotides to form double-stranded nucleic acids without the use of polymerase enzymes.

[0047] In some embodiments, the recombinant nucleic acids in the sublibrary also include nucleic acid sequences encoding protein tags so that the peptides encoded by the recombinant nucleic acids can be isolated using a binder for the protein tags. For example, preferred protein tags include the FLAG tag (having the sequence motif DYKDDDDK), the Myc tag (having the sequence motif EQKLISEEDL), and the HA- tag. In some embodiments, the protein tags may be repeated to enhance the signal or increase detection (e.g., three repetitions of the FLAG tag (3X FLAG)).

[0048] Several random sequence cassettes inserted into the recombinant nucleic acids of the sublibrary are intended to introduce frameshifts, nonsense mutations, and sequences that destabilize the structure of the peptide encoded by the recombinant nucleic acid. Therefore, in some embodiments, the inventors intend to test the recombinant nucleic acids of the sublibrary in vitro so that any recombinant nucleic acids encoding unstable or misfolded peptides can be removed from the library. For example, V H -CDR3 sublibrary or V L Each of the recombinant nucleic acids in the sublibrary independently of the CDR sequence is an immunoglobulin V H3 domains or V L The binding affinity of Staphylococcus aureus protein A or Finegoldia magna protein L to the structured epitope of the (Vκ) domain can be tested.

[0049] Any suitable method for screening recombinant nucleic acids by their binding affinity to protein A or protein L is contemplated. In an exemplary embodiment, recombinant nucleic acids from a sublibrary are transcribed into mRNA by in vitro transcription, and the 3' end of the mRNA is bound (covalently) to puromycin. The puromycin-bound mRNA is transcribed in vitro so that the peptide transcribed from the puromycin binds to the mRNA via puromycin. The peptide is then contacted with protein A or protein L to identify peptides that efficiently bind to protein A or protein L. Preferably, 10 -6 M or less, preferably 10 -7 K below M D A peptide that has affinity for protein A or protein L 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 produced via in vitro reverse transcription of puromycin and mRNA bound to the peptide. Next, the cDNA of the isolated peptide thus produced is V H -CDR3 sublibrary or V L It may be inserted as a random sequence cassette for generating selected recombinant nucleic acids in the sublibrary. Alternatively, it is also intended that the recombinant nucleic acids in the sublibrary may exist in the form of mRNA pre-bound to a puromycin molecule, by option, so that an in vitro transcription step (in DNA format) for recombinant nucleic acids is not required.

[0050] Building an scFv library from a sublibrary The inventors further intend to form recombinant scFv nucleic acids by recombining at least two recombinant nucleic acids (members) from the sublibrary. In a preferred embodiment, each of the at least two recombinant nucleic acids (members) is selected from a different sublibrary. For example, one recombinant nucleic acid is V H -CDR1 / 2 sublibrary, multiple V H -CDR3 sublibrary, and V L Each of the sublibraries can be selected. Regarding another example, one recombinant nucleic acid is V H -CDR1 / 2 sublibrary, multiple V H -CDR3 sublibrary, and V L A selection may be made from each of two of the sublibraries. Preferably, at least one, more preferably all, of the recombinant nucleic acids selected from the sublibraries are pre-selected by affinity binding screening as described above.

[0051] Most typically, recombinant scFv nucleic acids can be constructed by recombining a portion of recombinant nucleic acids derived from a sublibrary. In this embodiment, the portion of recombinant nucleic acid includes a random sequence cassette inserted into the recombinant nucleic acid. Therefore, for example, as a first step, V H -Some of the recombinant nucleic acids in the CDR1 / 2 sublibrary are [ka] (Randomly arranged cassettes are underlined), preferably, [ka] more, [ka] It may also be V H -Some of the recombinant nucleic acids in the CDR3 sublibrary are [ka] (Randomly arranged cassettes are underlined), preferably, [ka] more, [ka] It may also be V H -CDR1 / 2 sublibrary and V H -A portion of recombinant nucleic acids derived from the CDR3 sublibrary is isolated (e.g., by PCR), recombined (e.g., fused via restriction-ligation, prepared via recombinant PCR, etc.), V H Domain-recombinant nucleic acids can be formed. Therefore, typically, V H Domain recombinant nucleic acids are [ka] (Randomly arranged cassettes are underlined) This is likely the structure. By arbitrary selection, V H Domain-recombinant nucleic acids may also contain nucleic acid sequences that encode protein tags (e.g., FLAG tags, Myc tags, HA tags, etc.) at their 3' end, as described above. In addition, V produced in this manner H Domain recombinant nucleic acids are V H As a domain library member, V H It can be placed within a domain library.

[0052] V formed in this way H Domain recombinant nucleic acids are further V L Recombinant scFv nucleic acids can be formed by recombination with recombinant nucleic acids from a sublibrary. Figure 5 shows one exemplary method for recombining sequences from a sublibrary. As shown, and typically, V H Part of the domain recombinant nucleic acid and V L A portion of the recombinant nucleic acids in the sublibrary is fused to the recombinant scFv nucleic acid. For example, V L The recombinant nucleic acids in the sublibrary are V HV H Some of the recombinant nucleic acids are [ka] (preferably without any nucleic acid encoding a protein tag at its 3' end) and V L Some of the recombinant nucleic acids in the sublibrary are [ka] It may also contain (a promoter and 5'-UTR). Therefore, a typical recombinant scFv nucleic acid is [ka] It is likely the structure of V. H Part of the domain recombinant nucleic acid and V L It is highly preferable that some of the recombinant nucleic acids in the sublibrary be placed in the same reading frame so as to encode a single polypeptide.

[0053] Preferably, V H Part of the domain recombinant nucleic acid and V L A portion of the recombinant nucleic acids in the sublibrary is fused via a nucleic acid encoding a linker (short peptide spacer fragment) between the two portions. Any suitable length and order of peptide sequences for the linker or spacer can be used. However, the length of the linker peptide is preferably 3 to 30 amino acids, preferably 5 to 20 amino acids, and more preferably 5 to 15 amino acids. For example, the inventors have found that glycine-rich sequences (e.g., gly-gly-ser-gly-gly, etc.) are suitable. H and V L It is intended to be used to enable the mobility of scFv across domains.

[0054] Optionally, recombinant scFv nucleic acids may also contain nucleic acid sequences encoding protein tags (e.g., FLAG tags, Myc tags, HA tags, etc.) at their 3' end, as described above. In addition, recombinant scFv nucleic acids thus constructed may be placed in an expression library as members of the expression library.

[0055] In some embodiments, the recombinant scFv nucleic acids thus formed are further screened and / or classified based on their binding affinity to one or more target ligands (e.g., cancer antigens, neoepitopes, etc.), stability, pH sensitivity, and / or interspecies cross-reactivity. For example, the stability of scFv peptides encoded by recombinant scFv nucleic acids can be analyzed by size exclusion chromatography, which measures the size of the peptide over time. In other examples, the pH sensitivity and binding affinity of scFv peptides encoded by recombinant scFv nucleic acids can be analyzed by contacting the scFv peptides with one or more ligands under different buffer conditions (pH, temperature, etc.).

[0056] With regard to the analysis and further isolation of desired recombinant scFv nucleic acids derived from expression libraries, the inventors intend that recombinant scFv nucleic acids may exist in the form of mRNA, and optionally, that the mRNA is pre-bound with a puromycin molecule at its 3' end. The puromycin-bound mRNA can then be transcribed in vitro so that the peptide transcribed from the puromycin binds to the mRNA via puromycin. The peptide is then contacted with one or more ligands under optionally different buffer conditions (pH, temperature, etc.). Preferably, at pH 5.0-8.0, preferably pH 6.0-8.0, more preferably pH 6.5-8.0 for 10 -6 M or less, preferably 10 -7 K below M DPeptides that have affinity for a ligand and bind to it are selected and isolated. Once peptides with high affinity for the ligand are isolated, the cDNA of the isolated peptide can be produced via in vitro reverse transcription of puromycin and mRNA bound to the peptide.

[0057] Furthermore, the cDNA thus prepared from an isolated peptide encoded by recombinant scFv nucleic acid can be transplanted and replaced in a portion of an immunoglobulin to form recombinant immunoglobulin or a fragment thereof. For example, the cDNA thus prepared can be fused with the backbone of the constant region of the immunoglobulin heavy chain so that the variable regions of the heavy and light chains of the immunoglobulin can be replaced with scFv formed by the isolated peptide. Alternatively, the inventors may also use the V of recombinant scFv nucleic acid. H Part (or V H (derived from recombinant nucleic acids) and V L The aim is to be able to transplant and replace a portion (or a portion derived from recombinant scFv nucleic acid) of an immunoglobulin to form a recombinant immunoglobulin or a fragment thereof. For example, the V portion of recombinant scFv nucleic acid. H Part (or V H (derived from recombinant nucleic acids) and V L The portion (or one derived from recombinant scFv nucleic acid) is fused with the backbone of either the heavy chain constant region or the light chain constant region of an immunoglobulin to form an immunoglobulin having a variable region specific to the desired ligand.

[0058] In these examples, immunoglobulins are intended to include any type of heavy chain or constant domain constituting different types of immunoglobulins (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) and any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). In addition, “antibodies” may include, but are not limited to, human antibodies, humanized antibodies, chimeric antibodies, monoclonal antibodies, and polyclonal antibodies. In this context, isolated V by the intended system and method H and V LIt should be noted that species-specific antibodies can be produced by transplanting the domain into the remainder of an antibody of a desired species (e.g., human). In another example, the cDNA thus produced can be fused with a nucleic acid encoding another portion of the immunoglobulin to form an immunoglobulin fragment. In this example, the immunoglobulin fragment is intended to be a Fab fragment, a Fab' fragment, F(ab')2, a disulfide bond Fv(sdFv), and Fv. The inventors further fused a portion of the cDNA thus produced with a nucleic acid encoding another portion of the immunoglobulin to form V H Segment and / or V L The intention is to be able to form any fragment that contains any of the segments.

[0059] Furthermore, the inventors intend that the scFv moiety can be used as a targeting entity for various protein and non-protein molecules. For example, the scFv moiety may be conjugated to an ALT-803 type molecule (typically as a chimeric protein) to form a TxM entity with specific targeting capabilities (see, for example, J Biol Chem. 2016 Nov 11;291(46):23869-23881). In another example, the scFv moiety may be conjugated to a carrier protein (e.g., albumin) to enable target-specific delivery of one or more drugs to specific locations in the tumor microenvironment when the drug is bound to the carrier.

[0060] The inventors further constructed sublibraries through targeted diversification of random sequences and / or pre-selected members of the sublibraries, thereby enabling the expression library to minimize the sacrifice of diversity by removing unstable sequences, unbound sequences, or misfolded sequences, resulting in approximately 10 12The aim is to achieve this level of complexity. Therefore, the above approach for constructing expression libraries is practical for screening binders / antibodies in small quantities while providing a meaningful size for sequence complexity. In addition, the above approach for constructing expression libraries simplifies the binder / antibody screening procedure. Traditionally, in vitro validation of any nucleic acid sequence encoding a binding domain (or motif) (e.g., a randomized sequence) has been performed by F ab It was necessary to convert the nucleic acid sequence to a domain, and then the binding affinity could be tested via a pull-down assay with the ligand of interest. The method presented herein involves converting the nucleic acid sequence to an F ab Without converting to domains, this enables in vitro validation of nucleic acid sequences encoding binding domains (or motifs) through ranking by affinity (e.g., Kd value), pH sensitivity, and interspecific cross-reactivity (e.g., via surface plasmon resonance assays). Furthermore, pre-selection of members from each library based on stability and sensitivity reduces the pool that will be tested in the library, allowing for more rapid and efficient identification of desired binder / scFv / antibody domains. Accordingly, we also use mRNA display technology, in which post-in vitro library members are screened against solid-bound antigens, to select high-affinity binders (e.g., nano and picomolar K) from a high-diversity pool. d A method is devised for isolating (having). Once the binders are identified, they are determined to have affinity and K as further described below. on / K off The properties can be further characterized by surface plasmon resonance spectroscopy. From another perspective, the intended system and method enable rapid detection of the binder and production of scFv or antibody in a process completely independent of the in vivo immune system. [Examples]

[0061] Examples To maximize diversity while maintaining efficiency, any suitable diversification scheme may be contemplated to identify the target diversification region(s). However, the inventors found that VH3 / Vk1 could be one of the good candidate regions for randomization among the various domains of immunoglobulin, with VH3 being considered the most stable and soluble VH domain, and the light chain Vk1 being stable and soluble. Therefore, VH3 / Vk1 randomization pairs are intended to be converted to full-size immunoglobulins more efficiently. Accordingly, the inventors developed a pre-selection strategy using the VH3 and Vk1 framework. Figure 1 shows one exemplary randomization strategy using the VH3 / Vk1 pair. The protein sequences of at least 14 immunoglobulin molecules specific to one antigen are compared and analyzed. The most stable and conserved sequence among the 14 immunoglobulin molecules is used as the framework, and the position of the variable sequence is analyzed and used as the randomized sequence and degree of randomization (e.g., fully random, partially random, etc.).

[0062] Based on this randomization strategy, the inventors further propose V H Domains CDR1, CDR2-n, CDR2-c (see Figure 2) and V H Targeted diversified sequences (randomized sequences, random oligos) were generated for the CDR3 domain (see Figure 3). H Domain CDR1, CDR2-n, CDR2-c, CDR3, and V L The process for producing recombinant scFv nucleic acids using random oligonucleotides of the domain CDR3 is described above and is also shown in the schematic diagram in Figure 4. The high-diversity library is constructed as exemplified in Figure 5 and is discussed in more detail above.

[0063] Using a targeted diversification scheme and method for producing recombinant scFv nucleic acids as described in Figures 1-5, the inventors prepared a highly diverse library from which recombinant α-B7-H4 801(α-B7-H4, clone number 801) Binder was isolated. Recombinant α-B7-H4 801 The stability of α-B7-H4 was determined by 15 minutes of analytical size exclusion chromatography to evaluate antibody degradation or deformation. As shown in Figure 6, 801 The eluate showed a single peak without significantly small peaks, which is the α-B7-H4 produced by the method described above. 801 This indicates that the binder was able to produce scFv or antibodies with high stability.

[0064] The inventors of this invention have identified recombinant α-B7-H4 801 However, it was found to contain substantially the same antibody components as other commercially available α-B7-H4 antibodies (Rituxan®, LEAF®). Recombinant α-B7-H4 801 Fragments of two commercially available α-B7-H4 antibodies (Rituxan® and LEAF®) were analyzed via capillary electrophoresis using sodium dodecyl sulfate (CE-SDS). As shown in Figure 7, recombinant α-B7-H4 801 CE-SDS separation of the antibody and two commercially available α-B7-H4 antibody fragments (Rituxan®, LEAF®) shows two prominent peaks, corresponding to the light chain (center peak) and the glycosylated heavy chain (right peak), respectively. The left peak indicates the position of the standard 10 kD marker for CE-SDS analysis.

[0065] The inventors further found that various recombinant α-B7-H4 antibodies can exhibit different binding characteristics (e.g., affinity, specificity, etc.) to their target ligands. Figure 8 shows two recombinant α-B7-H4 antibodies, α-B7-H4, being tested for binding to B7-H4-expressing 293T cells, as measured by mean fluorescence intensity (MFI). 801 and α-B7-H4 817 This shows that the result is α-B7-H4 801 The antibody is α-B7-H4 817The study showed higher binding affinity to B7-H4 expressing 293T cells compared to the antibody, indicating that separately randomized CDR domains can result in different binding affinities to ligands. The far right panel shows a control experiment using nonspecific human IgG1 (hIgG1).

[0066] Recombinant α-B7-H4 antibodies were further tested using flow cytometry to determine specific and efficient binding to the ligand (B7-H4) expressed on antigen-presenting cells (APCs). As shown in Figure 9, the recombinant α-B7-H4 antibody was able to specifically bind to the B7-H4 ligand (separating the peak out from nonspecific isotype binding), indicating that the recombinant α-B7-H4 antibody is fully functional.

[0067] The inventors also proposed B7-H4(scFv B7-H4 801 ) scFv peptide and scFv B7-H4 801 Along with recombinant α-B7-H4 antibody (IgG α-B7-H4) produced using the same scFv peptide. 801 ) were found to be functionally compatible using a surface plasmon resonance assay. In this assay, Flag-tagged scFv B7-H4 801 It is immobilized on the surface via an α-Flag biotinylated antibody conjugated to surface-bound neutraavidin. Next, the immobilized scFv B7-H4 801 The peptide is contacted with an analyte containing B7-H4. A similar assay was performed with an α-B7-H4 antibody. As shown in Figure 10 and Table 3, scFv B7-H4 801 and IgG α-B7-H4 801 These exhibit substantially similar affinity and binding characteristics to B7-H4, indicating their functional compatibility. Furthermore, since the binding affinity of in vitro translated peptides (scFv) can be measured directly without transplanting the peptide into an antibody backbone, a greater number of recombinant scFv nucleic acids in the expression library can be efficiently screened.

[0068] [Table 4]

[0069] V H CDR1~3 and V L Among several scFv peptides for B7-H4 having various random sequence cassettes in the CDR3, the inventors tested whether similarity in a particular domain (a particular random sequence cassette) could cause the scFv peptides to have similar binding properties to the ligand. Five scFv peptides (801, 802, 905, 906, and 817) were tested for their binding affinity to B7-H4. As shown in Table 4, four of these scFv peptides (clones 801, 802, 905, and 906) have similar CDR3 sequences. H The four scFv peptides, which have similar random sequence cassettes in CDR3, showed similar binding affinity to B7-H4 at both 25°C and 37°C (as shown in Table 5), indicating that at least among the scFv peptides for B7-H4, V H This suggests that the sequence in CDR3 may be important for ligand binding.

[0070] [Table 5]

[0071] [Table 6]

[0072] The inventors also prepared several scFv peptides that bind to interleukin-8 (IL-8) (scFv IL-8) using sublibraries and expression libraries, and tested their affinity for IL-8 under various conditions (temperature and pH). Exemplary scFv 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 the CDR3 sequence, and clones 49-19, 49-37, and 49-25 are similar V H It contains the CDR3 sequence. In addition, clones 49-3 and 43-2 have similar V H It contains the CDR3 sequence. In contrast to the scFv peptide for B7-H4, the inventors have found that the binding affinity of the scFv IL-8 peptide is V H We found that the similarity in random sequences in CDR3 is not always critically dependent. For example, clones 49-18, 49-37, and 49-25 are similar V H While containing the CDR3 sequence, the binding affinity of those sequences (K D X10 -9 The unit measured in M ​​is 0.894 x 10 -9 M and 25X10 -9 It fluctuates within M.

[0073] [Table 7]

[0074] The inventors further tested whether scFv IL-8 could neutralize the effects of IL-8 by efficiently capturing it, by measuring neutrophil size. Generally, neutrophils enlarge when stimulated by IL-8 (e.g., they have a larger diameter) (as shown in Figure 11). The inventors investigated such IL-8 action on neutrophil enlargement using a recombinant α-IL-8 antibody (mAb αIL-8). 201As shown in Figure 12, (upper left graph) or several scFv IL-8 peptides (αIL-8 #2 , αIL-8 49-3 , αIL-8 49-10 As shown in Figure 12, we found that the scFv IL-8 peptide (in the lower graph) could be largely eliminated by its addition, which indicates that the scFv IL-8 peptide was able to efficiently neutralize the effects of IL-8 by binding to free IL-8 in the culture medium.

[0075] IL-8 is a neutrophil chemotactic factor that causes neutrophils to migrate toward IL-8 release sites (e.g., sites of infection). To evaluate the functional effects of the scFv IL-8 peptide, neutrophils were placed at the bottom of an insert with a porous membrane and placed in a medium containing various concentrations of IL-8 so that IL-8-attracted neutrophils could migrate from the insert to the culture medium through the porous membrane. As shown in Figure 13, increasing the IL-8 concentration in the culture medium increased the number of migrating neutrophils. Interestingly, such IL-8 activity is attributed to the scFv IL-8 peptide (αIL-8). 43-2 ) or recombinant IL-8 antibody derived from scFv IL-8 peptide (mAb αIL-8 201 The addition of ) almost completely eliminated it.

[0076] Figure 14 shows further experimental data for various scFv isolated using the mRNA display library presented herein. More specifically, each data point represents an scFv for the target shown below, and the affinity value for each scFv was determined. As can be seen, the (same) library yielded multiple high-affinity binders for various different targets, all binders having affinity ranges of micro-M or less, and many of nano-M or less. Furthermore, we also investigated whether the affinity of scFv can be maintained when CDRs are transplanted onto human IgG. Figure 15 shows exemplary results from 29 CDR transplantation experiments for selected scFvs transplanted onto human IgG1 scaffolds. As can be seen from the results in Figure 15, the humanized IgG1 antibody retained high specificity and affinity (typically within a single order of magnitude).

[0077] Creation of recombinant entities using expression libraries V H Domain-recombinant nucleic acids and V LIt is further intended that recombinant scFv nucleic acids, or recombinant nucleic acids encoding one or more antibodies (e.g., IgG, IGM, IgE, IgA, etc.), or fragments thereof, formed by the recombination of recombinant nucleic acids in a sublibrary, may be further inserted into an expression vector of a recombinant entity (e.g., bacteria, yeast, virus) so that the recombinant scFv fragment can be produced by the recombinant entity or cells infected with the recombinant entity. Any suitable recombinant entity capable of carrying and / or expressing recombinant nucleic acids encoding the recombinant scFv fragment is intended. For example, any suitable virus may be used as the recombinant entity, e.g., adenovirus, adeno-associated virus, alphavirus, herpesvirus, lentivirus, etc. However, adenovirus is particularly preferred. Furthermore, it is even more preferred that the virus is replication-deficient and non-immunogenic, which is typically realized by targeted deletion of selected viral proteins (e.g., E1, E3 proteins). Such desired properties can be further enhanced by deleting E2b gene function, and high titers of recombinant viruses can be achieved using genetically modified human 293 cells, as recently reported (e.g., J Virol. 1998 Feb;72(2):926-933). Therefore, we intend for a single desirable viral vector to contain a recombinant adenovirus genome in which the E2b gene is deleted or non-functional.

[0078] Alternatively, the recombinant entity may be a bacterium, and the expression vector may be a genetically modified bacterium that expresses endotoxins at levels low enough not to cause an endotoxin reaction in human cells, and / or a bacterial vector that may be insufficient to induce CD14-mediated sepsis when introduced into the human body. An example of a bacterial strain with modified lipopolysaccharide is ClearColi® BL21(DE3) electrocompetent cell. This bacterial strain is BL21 with the genotype F-ompT hsdSB(rB-mB-)gal dcm lon λ(DE3[lacI lacUV5-T7 gene 1 ind1 sam7 nin5])msbA148 ΔgutQΔkdsD ΔlpxLΔlpxMΔpagPΔlpxPΔeptA. In this regard, several specific deletion mutations (ΔgutQ ΔkdsD ΔlpxL ΔlpxMΔpagPΔlpxPΔeptA) are lipid IV of LPS. A While it codes for a modification to LPS, it should be understood that one additional compensatory mutation (msbA148) allows cells to survive in the presence of LPS precursor lipid IVA. These mutations result in the deletion of oligosaccharide chains from LPS. More specifically, two of the six acyl chains are deleted. The six acyl chains of LPS are triggers recognized by Toll-like receptor 4 (TLR4), which is a complex with myeloid differentiation factor 2 (MD-2), leading to NF-κB activation and the production of inflammatory cytokines. Lipid VI contains only four acyl chains. A It is not recognized by TLR4 and therefore does not trigger an endotoxin reaction. Although the electrocompetent BL21 bacterium is described as an example, the inventors intend that the genetically modified bacteria may also be chemically competent bacteria. Alternatively, the recombinant entity may be yeast, and the expression vector may also be a yeast vector that can be expressed in yeast (preferably Saccharomyces cerevisiae (e.g., GI-400 series recombinant immunotherapy yeast strains)).

[0079] The inventors may increase the diversity of therapeutically effective recombinant entities by constructing a set of recombinant entities (e.g., recombinant viruses) using multiple recombinant scFv nucleic acids and / or recombinant nucleic acids encoding one or more antibodies (e.g., IgG, IgM, IgE, IgA, etc.). Preferably, the recombinant nucleic acids encoding multiple recombinant scFv and / or one or more antibodies (e.g., IgG, IgM, IgE, IgA, etc.) may be selected based on the affinity and / or binding properties (e.g., binding kinetics, etc.) of the scFv fragments or antibodies to an antigen, so that the top 30%, top 20%, top 10%, or 5% of the scFv fragments or antibodies with the highest binding affinity or other binding properties can be selected from a pool of scFv fragments or antibodies. In some embodiments, such a selection process may include the selection and enrichment of high-pass fragments through multiple rounds of selection. For example, in some embodiments, the top 30% of scFV fragments or antibodies with the highest binding affinity may be selected in the first round of selection, and the top 50% of the top 30% of scFV fragments or antibodies with the highest binding affinity (from the first round) may be selected in the second round of selection, and so on. Any appropriate number of rounds and pass rates (e.g., top 30%, top 20%, etc.) of selection may be used, but it is preferable that the final set of scFV fragments or antibodies may constitute up to 30%, top 20%, top 10%, or 5% of the scFV fragments with the highest binding affinity in the entire pool of scFV fragments or antibodies.

[0080] The recombinant scFv nucleic acids obtained in this manner, and / or sets of recombinant nucleic acids encoding one or more antibodies (e.g., IgG, IgM, IgE, IgA, etc.), can be used to create a heterogeneous pool of recombinant entities (e.g., recombinant viruses) that can further produce multiple different scFv fragments and / or antibodies that bind to the same antigen. While we do not wish to be bound by any particular theory, we anticipate that such an approach may increase the opportunity to identify scFv fragments and / or antibodies that can bind most effectively to the antigen by increasing the pool of high-affinity candidate scFv fragments. Conversely, the recombinant scFv fragment or antibody with the highest binding affinity may not be the most therapeutically effective scFv fragment or antibody due to many in vivo variables (e.g., slight individual structural differences in the antigen between patients, different binding conditions (e.g., pH, other environmental interferences, etc.)), or because the recombinant scFv fragment or antibody with the highest binding affinity may have kinetic properties undesirable as a therapeutic antibody. Therefore, by creating a heterogeneous pool of recombinant entities using sets of nucleic acids encoding various scFv fragments or antibodies, there is an opportunity to identify therapeutically effective scFv fragments (even if they do not have the highest binding affinity to the antigen). In addition, a heterogeneous pool of antibodies (created from a heterogeneous pool of recombinant entities) that bind to the same antigen may increase the effectiveness of targeting the antigen in vivo compared to a homogeneous pool of antibodies that may simultaneously lose effectiveness under certain in vivo conditions. Conversely, we anticipate that a heterogeneous pool of recombinant entities (particularly recombinant viruses carrying recombinant nucleic acids encoding various scFv fragments or antibodies targeting the same antigen) may be more therapeutically beneficial and / or effective in treating patients with tumors.

[0081] In some embodiments, the expression vector may include a nucleic acid segment encoding a signaling peptide for extracellular secretion, so that the produced recombinant scFv or antibody can be secreted from the cell. Any suitable signaling peptide is intended, but exemplary signaling peptides may comprise 5 to 30 amino acids having a positively charged N-terminal region (n-region), a hydrophobic central region (h-region), and a neutral polar C-terminal region (c-region), the amino acids of 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 scFv (optionally via a shorter linker, e.g., a nucleic acid encoding a glycine-rich linker).

[0082] Alternatively and / or in addition, the expression vector may further include one or more elements that can induce or increase an immune response against a tumor and / or increase the activity of the constructed scFv fragment. Therefore, in some embodiments, the expression vector may include another nucleic acid segment encoding a neoantigen, tumor-associated antigen, or tumor-specific patient-specific neoepitope, a co-stimulatory molecule, an immunostimulatory cytokine, a recombinant immunoglobulin protein complex, and / or a checkpoint inhibitor. With respect to this cytokine, any suitable cytokine capable of modulating the immune response (e.g., increasing or decreasing T cell activity) is intended. Therefore, potential co-stimulatory molecules include 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, this cytokine may be an IL-15 superagonist (IL-15N72D) and / or an IL-15 superagonist / IL-15RαSushi-Fc fusion complex (e.g., ALT-803) bound to at least one of IL-7, IL-15, IL-18, IL-21, and IL-22, or preferably both IL-7 and IL-21. Potential co-stimulatory molecules include 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 CTLA-4 (especially CD8). + In the case of cells, PD-1 (especially CD4 + In the case of cells, examples include antibodies or binding molecules against the TIM1 receptor, 2B4, and CD160, such as ipilimumab and nivolumab.

[0083] In some embodiments, recombinant entities can be used to produce scFv fragments and / or antibodies in vitro. For example, recombinant bacteria or recombinant yeast can be induced and / or cultured to express scFv proteins, and the thus expressed scFv fragment proteins can be further purified and / or isolated by any suitable method (e.g., affinity-binding purification) for further use. In other embodiments, recombinant entities can be used to infect mammalian cells in vivo and / or ex vivo. For example, a recombinant virus having recombinant nucleic acid encoding scFv fragments 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. In such examples, the mammalian cell could be any suitable mammalian cell capable of producing a protein from an exogenous nucleic acid that secretes a recombinant scFv fragment, and such mammalian cells could be any established cell lineage (human or non-human, e.g., HEK-293 cells, CHO cells, etc.) or autologous cells obtained from a patient with a tumor (e.g., autologous B cells isolated and / or expanded in ex vivo).

[0084] Alternatively, a recombinant virus having recombinant nucleic acid encoding an scFc fragment and / or antibody may be administered to a human or mammal (having a tumor) so as to produce and secrete the scFc fragment and / or antibody in vivo. In such embodiments, the recombinant virus may be formulated on any pharmaceutically acceptable carrier, preferably with a viral titer of 10 per dose unit. 4 ~10 12It can be formulated as a sterile injection composition consisting of individual virus particles. However, alternative formulations are also considered suitable for use as described herein, and all known routes and modes of administration are intended herein. As used herein, the term “administer” a recombinant virus formulation refers to both direct and indirect administration of the recombinant virus formulation, where direct administration is typically performed by a healthcare worker (e.g., physician, nurse, etc.), and indirect administration includes the step of providing or making available the recombinant virus formulation to a healthcare worker for direct administration (e.g., by injection, infusion, oral delivery, local delivery, etc.).

[0085] In some embodiments, the recombinant virus preparation is administered by systemic injection, such as subcutaneous, subdermal, or intravenous injection. In other embodiments, where systemic injection may not be efficient (for example, in the case of brain tumors), the recombinant virus preparation is intended to be administered by intratumoral injection.

[0086] With regard to the dose and schedule of recombinant virus preparation administration, it is intended that this dose and / or schedule may vary depending on the type, size, and location of the tumor, the patient's health status (including, e.g., age, sex, etc.), and any other relevant conditions. Although it may vary, this dose and schedule can be selected and adjusted so that the recombinant virus does not cause any significant toxic effects on the host's normal cells, and is still effective in inducing the production of recombinant scFv fragments to thus treat the tumor. For example, the intended dose of recombinant virus preparation is at least 10 6 10 virus particles / day, or at least 10 8 10 virus particles / day, or at least 10 10 10 virus particles / day, or at least 10 11This is 10

[0087] It will be apparent to those skilled in the art that many variations are possible beyond those already described, without departing from the inventive concept of this specification. Therefore, the subject matter of the present invention is not limited beyond the appended claims. Furthermore, in interpreting both this specification and the claims, all terms should be interpreted in the broadest possible way in the context. In particular, the terms “including” and “including” should be interpreted as referring to an element, component, or process in a non-exclusive manner, indicating that the referenced element, component, or process may exist, be utilized, or be combined with other elements, components, or processes not explicitly referenced. As used throughout this specification and the subsequent claims, the meanings of “a,” “an,” and “the” include multiple references unless the context clearly indicates otherwise. Similarly, as used herein, the meaning of “in” includes “in” and “on” unless the context clearly indicates otherwise. Where a claim in this specification refers to at least one selected from the group consisting of A, B, C, ..., and N, the text should be interpreted as requiring only one element from the group, rather than A+N or B+N, etc.

Claims

1. It is a recombinant virus, Recombinant nucleic acids comprising members of an expression library encoding a separate antibody or antibody fragment, The members of the expression library are (1) V Ｈ - CDR1 / 2 sublibrary, (2) multiple V Ｈ -CDR3 sublibrary, and (3)V Ｌ This is a process of creating or providing a sublibrary, where each of the sublibraries (1) to (3) includes multiple members. Each member of the sublibrary comprises at least one random cassette having a plurality of degenerate base positions, and is prepared or provided in the process of: The aforementioned V Ｈ - CDR1 / 2 sublibrary, the plurality of V Ｈ - CDR3 sublibrary, and the V Ｌ A step of rearranging at least a portion of at least two members of a sublibrary to form the expression library member in the expression library. Recombinant viruses produced by [company name / organization name].

2. The recombinant virus according to claim 1, which is a genetically modified, low-immunogenic virus.

3. The recombinant virus according to claim 2, which is human adenovirus serotype 5 having a mutation in at least one of the following genes: E1A, E1B, E2B, and E3.

4. Said V Ｈ - A plurality of members of the CDR1 / 2 sublibrary are V Ｈ A part of CDR1 and V Ｈ The recombinant virus according to any one of claims 1 to 3, comprising a random cassette corresponding to at least one of a part of CDR2.

5. The aforementioned V Ｈ - Multiple members of the CDR1 / 2 sublibrary, V Ｈ At least a portion of CDR1 and V Ｈ A recombinant virus according to any one of claims 1 to 4, comprising a plurality of random cassettes corresponding to a portion of CDR2.

6. The aforementioned V Ｈ - Multiple members of the CDR1 / 2 sublibrary, V Ｈ The recombinant virus according to claim 4, comprising a plurality of random cassettes corresponding to at least a portion of CDR2.

7. The aforementioned V Ｈ - Multiple members of the CDR3 sublibrary, V Ｈ A recombinant virus according to any one of claims 1 to 6, comprising a random cassette corresponding to at least a portion of CDR3.

8. The aforementioned V Ｈ - The recombinant virus according to any one of claims 1 to 7, wherein at least two random cassettes of members of the CDR3 sublibrary encode peptides of varying lengths.

9. The recombinant virus according to claim 8, wherein the peptide has a length in the range of 10 to 20 amino acids.

10. The recombinant virus according to any one of claims 1 to 9, wherein the plurality of members of the sublibrary have a common sequence.

11. The aforementioned V Ｌ The aforementioned multiple members of the sublibrary are V Ｌ A recombinant virus according to any one of claims 1 to 10, wherein a portion of the CDR3 includes a random cassette.

12. The above rearrangement step is V Ｈ - At least a portion of the members of the CDR1 / 2 sublibrary and the plurality of V Ｈ - Isolate one of the CDR3 sublibraries and fuse them together to form V Ｈ V in domain libraries Ｈ A step in forming a domain library member, the V Ｈ Domain libraries are multiple V Ｈ A recombinant virus according to any one of claims 1 to 11, comprising the step of forming a domain library member.

13. The member of the expression library is the V Ｌ Isolate at least a portion of the members of the sublibrary, and V Ｌ A portion of the aforementioned members of the sublibrary, and the V Ｈ The recombinant virus according to claim 12, which is produced by fusing one of the domain library members with the expression library member.

14. The above rearrangement step is V Ｈ - At least a portion of the members of the CDR1 / 2 sublibrary and the plurality of V Ｈ - A recombinant virus according to any one of claims 1 to 13, comprising the step of isolating one of the CDR3 sublibraries and fusing them together to form a first group of expression library members.

15. The recombinant virus according to any one of claims 1 to 14, wherein the recombinant nucleic acid further comprises a nucleic acid fragment encoding a signaling peptide that promotes the secretion of the separate antibody or antibody fragment.

16. The recombinant virus according to any one of claims 1 to 15, wherein the separate antibody or antibody fragment comprises scFv.

17. The recombinant virus according to claim 17, further comprising the step of subcloning the expression library member to construct an IgG1 having the scFv.

18. The recombinant virus according to any one of claims 1 to 17, wherein the random cassette is prepared using oligonucleotides selected from SEQ ID NOs: 1 to 25.

19. (1) V Ｈ - CDR1 / 2 sublibrary, (2) multiple V Ｈ -CDR3 sublibrary, and (3)V Ｌ A process of creating or providing a sublibrary, wherein each of the sublibraries (1) to (3) includes multiple members; A step in which each member of the sublibrary includes at least one random cassette having a plurality of degenerate base positions; The aforementioned V Ｈ -CDR1 / 2 sublibrary, the plurality of VH-CDR3 sublibraries, and the V Ｌ A step of rearranging at least a portion of at least two members of a sublibrary to form an expression library member in an expression library, wherein the expression library comprises a plurality of expression library members, and each expression library member encodes a separate antibody or antibody fragment; and A process for preparing a recombinant viral vector containing at least one expression library member. A method for producing recombinant antibodies, including [the specified substance].

20. The method according to claim 19, wherein the recombinant viral vector is derived from a genetically modified low immunogenic virus.

21. The method according to claim 20, wherein the genetically modified low immunogenic virus is human adenovirus serotype 5 having a mutation in at least one of the following genes: E1A, E1B, E2B, E3.

22. The aforementioned V Ｈ - Multiple members of the CDR1 / 2 sublibrary, V Ｈ Part of CDR1 and V Ｈ The method according to any one of claims 19 to 21, comprising a random cassette corresponding to at least one of the CDR2.

23. The aforementioned V Ｈ - Multiple members of the CDR1 / 2 sublibrary, V Ｈ At least a portion of CDR1 and V Ｈ The method according to any one of claims 19 to 21, comprising a plurality of random cassettes corresponding to a portion of CDR2.

24. The aforementioned V Ｈ - Multiple members of the CDR1 / 2 sublibrary, V Ｈ The method according to claim 23, comprising a plurality of random cassettes corresponding to at least a portion of CDR2.

25. The aforementioned V Ｈ - Multiple members of the CDR3 sublibrary, V Ｈ The method according to any one of claims 19 to 24, comprising a random cassette corresponding to at least a portion of the CDR3.

26. The aforementioned V Ｈ - The method according to any one of claims 19 to 25, wherein at least two random cassettes of members of the CDR3 sublibrary encode peptides having different lengths.

27. The method according to claim 26, wherein the peptide has a length in the range of 10 to 20 amino acids.

28. The aforementioned V Ｌ The aforementioned multiple members of the sublibrary are V Ｌ The method according to any one of claims 19 to 27, wherein a portion of the CDR3 includes a random cassette.

29. The method according to any one of claims 19 to 28, wherein the plurality of members of the sublibrary have a common sequence.

30. The method according to any one of claims 19 to 29, wherein each of the expression library members includes a plurality of the random cassettes.

31. The rearrangement process is as described above V Ｈ - At least some of the members of the CDR1 / 2 sublibrary and the plurality of V Ｈ - Isolate one of the CDR3 sublibraries and fuse it together to create V Ｈ V in domain libraries Ｈ The process includes forming a domain library member, and the V Ｈ Domain libraries, multiple V Ｈ The method according to any one of claims 19 to 30, including a domain library member.

32. The aforementioned V Ｌ Isolate at least a portion of the members of the sublibrary, and V Ｌ Some of the members of the sublibrary are V Ｈ The method according to claim 31, further comprising the step of fusing with one of the domain libraries to form the expression library member.

33. The aforementioned V Ｌ The method according to claim 32, wherein some members of the sublibrary are joined via an array that codes for a linker.

34. The method according to claim 33, wherein the linker is a glycine-rich peptide.

35. The rearrangement process is as described above V Ｈ - At least some of the members of the CDR1 / 2 sublibrary and the plurality of V Ｈ - The method according to any one of claims 19 to 34, comprising the step of isolating one of the CDR3 sublibraries and fusing them together to form a first group of expression library members.

36. A second group of expression library members, wherein the second group is V Ｌ The method according to claim 35, further comprising the second group which includes at least a portion of the members of the sublibrary.

37. The method according to any one of claims 19 to 36, wherein the separate antibody or antibody fragment comprises scFv.

38. The method according to claim 37, further comprising the step of subcloning the expression library member to construct an IgG1 having the scFv.

39. Subcloning one member from each of the first and second groups described above, recombinant V Ｈ and V Ｌ The method according to claim 36, further comprising the step of forming an IgG1 having a domain.

40. The method according to any one of claims 19 to 39, further comprising the step of selecting a subset of the expression library members based on at least one of affinity for a ligand, pH sensitivity, and interspecific cross-reactivity.

41. The method according to claim 40, further comprising the step of selecting a subset of the expression library members that encode scFvs that bind to the ligand with a Kd of less than 50 nM.

42. The method according to any one of claims 19 to 41, wherein the random cassette is prepared using an oligonucleotide selected from SEQ ID NOs: 1 to 25.

43. The steps of transcribing the aforementioned expression library members into mRNA fragments, and The step of binding a puromycin molecule to the 3' end of the mRNA fragment. The method according to any one of claims 19 to 42, further comprising:

44. The method according to any one of claims 19 to 43, further comprising the step of contacting a recombinant virus having the recombinant viral vector with a mammalian cell.

45. The method according to any one of claims 19 to 44, wherein the contact step includes the step of administering the recombinant virus to a mammal.

46. The method according to any one of claims 1 to 45, wherein the mammalian cells are autologous cells of a patient having a tumor, and the contact step includes a step of co-incubating the autologous cells and the mammalian cells ex vivo.

47. Use of the composition according to any one of claims 1 to 18 for producing a recombinant antibody for therapeutic purposes against cancer neoepitopes.

48. A method for isolating a high-affinity binder having an affinity of 100 nM or less for an antigen, A method comprising the step of contacting the antigen with a composition constructed by any one of claims 19 to 46.

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