(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`(19) World Intellectual Property
`Organization
`International Bureau
`
`(43) International Publication Date
`3 November 2016 (03.11.2016)
`
`WIPOI PCT
`
`\9
`
`(10) International Publication Number
`
`WO 2016/173719 A1
`
`(51)
`
`International Patent Classification:
`C07K 16/00 (2006.01)
`C07K 16/40 (2006.01)
`
`(21)
`
`International Application Number:
`
`PCT/EP2016/000701
`
`(81)
`
`(74)
`
`Agent: LAHRTZ, Fritz; Isenbruck Bosl Horschler LLP,
`Patentanwalte, Prinzregentenstrasse 68, 81675 Mijnchen
`(DE).
`
`(22)
`
`International Filing Date:
`
`(25)
`
`(26)
`
`(30)
`
`(71)
`
`(72)
`
`Filing Language:
`
`Publication Language:
`
`29 April 2016 (29.04.2016)
`
`English
`
`English
`
`Priority Data:
`15001304.3
`
`30 April 2015 (30.04.2015)
`
`EP
`
`Applicants: ABCHECK S.R.0. [CZ/CZ]; Vedeckotech-
`nicky Park Plzen, Teslova 3, 301 00 Plzen (CZ). DIS-
`TRIBUTED BIO, INC. [US/US]; 660 4th Street, Suite
`491, San Francisco, CA 94107 (US).
`
`Inventors: GLANVILLE, Jacob, E., Gunn; 414 Lake
`Street, Apt
`1,
`San
`Francisco, CA 94118
`(US).
`MOLKENTHIN, Vera; Auf der Tralh 19, 92723 Tannes-
`berg (DE). GRIEP, Remko, Albert; Druzstevni 1680/12,
`30100 Plzen (CZ). TRAD, Ahmed; Nepomucka 156,
`32600 Plzen (CZ). MILOVNIK. Peter; K Male IIomolce
`30, 30100 Plzen (CZ). LANG, Volker; Kapellenweg 13a,
`85283 Wolnzach (DE).
`
`Designated States (unless otherwise indicated, for every
`kind of national protection available): AE, AG, AL, AM,
`AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY,
`BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM,
`DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT,
`HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KN, KP, KR,
`KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, MG,
`MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM,
`PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC,
`SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN,
`TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW.
`
`(84)
`
`Designated States (unless otherwise indicated, for every
`kind of regional protection available): ARIPO (BW, GH,
`GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ,
`TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU,
`TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE,
`DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU,
`LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK,
`SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ,
`GW. KM, ML, MR, NE, SN, TD, TG).
`Published:
`
`with international search report (Art. 21(3))
`
`(54) Title: METHOD FOR MASS HUMANIZATION OF RABBIT ANTIBODIES
`
`(57) Abstract: The present invention relates to a method for producing a population of 20 or more nucleic acids, each encoding at
`least one protein comprising at least one immunoglobulin variable domain having a rabbit-derived CDR3 amino acid sequence em-
`bedded in essentially human framework sequences, as well as to a population of nucleic acids and a population of proteins relates
`thereto and uses thereof.
`
`
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`PCT/EP2016/000701
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`Method for mass humanization of rabbit antibodies
`
`The present invention relates to a method for producing a population of nucleic
`
`acids encoding at
`
`least one protein comprising at
`
`least one immunoglobulin
`
`variable domain having a rabbit-derived CDR3 amino acid sequence embedded in
`
`essentially human framework sequences, as well as to a population of nucleic
`
`acids and a population of proteins relates thereto and uses thereof.
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`Inseparably connected with the advent of antibodies for human therapy are
`
`strategies to generate sequences that are not recognized as foreign by the human
`
`immune system. Up to the present animals are used to induce the generation of
`
`target specific antibodies in vivo and even the largest and most advanced in vitro
`
`generated libraries of human antibodies have not fully replaced the B—cells of
`
`immunized animals as source of antibodies for therapeutic applications, The
`
`continuous use of animal—derived antibodies raised a vivid and persistent interest
`
`in humanization strategies to transform a non-human antibody into a safe drug for
`
`human therapy.
`
`Jones et al. (Peter T. Jones, Paul H. Dear, Jefferson Foote, Michael S. Neuberger
`
`and Greg Winter, Nature 321: 522, 1986) published the humanization of a mouse
`
`antibody by CDR grafting nearly three decades ago. Riechmann et al.
`
`(Lutz
`
`Riechmann, Michael Clark, Herman Waldmann and Greg Winter, Nature 332: 323,
`
`1988), used the method in 1988 to humanize Campath® (Alemtuzumab), the first
`
`humanized antibody applied for therapeutic use. Since that time, developing and
`
`refining methods to predict required mutations in framework regions and CDRs
`
`that are essential to retain affinity and binding specificity, are subject of numerous
`
`publications, pioneered by Carter et al. (Paul Carter, Len Presta, Cornelia M.
`
`Gorman, Joh B. B. Ridgway, Dennis Henner, Wai Lee T. Wong, Ann M. Rowland,
`
`Claire Kotts, Monique E. Carver and Michael Shepard, PNAS 89: 4285, 1992) who
`
`humanized Herceptin® (Trastuzumab).
`
`Despite the tremendous gain of knowledge and improvement of antibody modeling
`
`software, CDR grafting is prone to turn into a lengthy procedure of trial and error,
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`WO 2016/173719
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`PCT/EP2016/000701
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`depending on sufficient structural information as well as on the experience and a
`lucky hand of the executing scientist.
`
`Humanizations by guided selections (Jane Osbourn, Maria Groves and Tristan
`
`Vaughan, Methods 36: 61, 2005) follow a different route. Libraries with either the
`
`VH or the VL of a non-human antibody paired with a set of their respective human
`
`counterparts are generated and subjected to selections. The human variable
`
`domains of the identified chimeric intermediates are combined, or again paired
`
`with a set of human counterparts and subjected to selections.
`
`in contrast to the
`
`CDR grafting, all traces of the non-human origin of antibodies humanized by
`
`guided selections are eliminated and resulting antibodies are to be called human
`rather than humanized.
`
`However,
`
`the method did not
`
`reach a similar
`
`level of awareness as the
`
`humanization via CDR grafting,
`
`lacking a comparable number of successful
`
`examples published in the literature.
`
`A common drawback of CDR grafting and humanization by guided selections is
`
`their limitation to one or a few antibodies at a time. CDR grafting is a highly
`
`individual process considering the structure of the respective antibody-antigen
`
`complex. Although the general approach of humanizations by guided selections
`
`allows a higher capacity, the library size is not infinite and limits the number of
`
`input candidates.
`
`This patent application describes a method that is applicable to all rabbit-derived
`
`antibodies and allows humanizations in high throughput and short time frames with
`
`reliable success rates.
`
`In one embodiment, the present invention relates to a method for producing a
`
`population of nucleic acids encoding at least one protein comprising at least one
`
`immunoglobulin variable domain having a rabbit-derived CDR3 amino acid
`
`sequence embedded in essentially human framework sequences, wherein the
`
`method comprises the following steps:
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`(a)
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`providing at least one nucleic acid encoding a rabbit-derived complementarity
`determining region 3 (CDR3) amino acid sequence or an amino acid
`
`sequence further encompassing 1, 2, or 3 amino acids N-terminal and/or C-
`
`terminal of the rabbit-derived CDR3 amino acid sequence,
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`WO 2016/173719
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`PCT/EP2016/000701
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`(b)
`
`generating a population of nucleic acids encoding at
`
`least one protein
`
`comprising at least one immunoglobulin variable domain having a rabbit
`
`CDR3 amino acid sequence of step (a) embedded in essentially human—
`
`framework sequences, wherein the human framework sequences comprise a
`
`first human framework region (FR1), a second human framework region
`
`, (FR2), a third human framework region (FR3), and a fourth human framework
`
`region (FR4),
`
`such that
`
`the FR1 and FR2 regions are interspaced by a complementarity
`
`determining region 1
`
`(CDR1),
`
`the FR2 and FR3 regions are interspaced by a
`
`complementarity determining region 2 (CDR2), and the FR3 and FR4 regions are
`
`interspaced by a rabbit-derived CDR3 amino acid sequence,
`
`wherein the nucleic acid sequences encoding the CDR1 and CDR2 amino acid
`
`sequences are diversified among the population of nucleic acids encoding at least
`
`one protein comprising at least one immunoglobulin variable domain,
`
`wherein each nucleic acid sequence encoding a CDR1 or CDR2 amino acid
`
`sequence is independently based
`
`i)
`
`on a nucleic acid sequence encoding a human CDR1 or CDR2, respectively,
`or
`
`ii)
`
`on a nucleic acid sequence encoding a rabbit CDR1 or CDR2, respectively,
`
`wherein at least some of the nucleic acid sequences encoding a CDR1 or CDR2
`
`amino acid sequence have been modified to encode at least one amino acid
`
`present in rabbit CDR1 or CDR2 amino acid sequences, respectively,
`
`in case of
`
`human CDR1 or CDR2, respectively, or to encode at least one amino acid present
`
`in human CDR1 or CDR2 amino acid sequences, respectively,
`
`in case of rabbit
`
`CDR1 or CDR2, respectively,
`
`and wherein the human FR1, FR2, FR3 and FR4 regions are human framework
`
`regions selected to provide a scaffold conducive for rabbit CDR3 amino acid
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`sequences,
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`with the proviso:
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`-
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`that the two C-terminal amino acids of FR2 are optionally non-human, and
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`PCT/EP2016/000701
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`-
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`that the two C-terminal amino acids of FR3 are optionally non—human.
`
`The present invention is in particular advantageous for mass humanization of
`rabbit antibodies, wherein a plurality of rabbit antibodies are to be'humanized
`
`efficiently. Preferably, the plurality of rabbit antibodies are humanized in parallel
`
`and/or without determining the amino acid sequences of the rabbit antibodies to be
`humanized.
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`Accordingly, in a preferred embodiment, the present invention relates to a method
`
`for producing a population of 20 or more nucleic acids, each encoding at least one
`
`protein comprising at least one immunoglobulin variable domain having a rabbit-
`
`derived CDR3 amino acid sequence embedded in essentially human framework
`
`sequences, wherein the nucleic acid sequences encoding the rabbit-derived CDR3
`
`amino acid sequences are diversified among the population of nucleic acids
`
`encoding at least one protein comprising at least one immunoglobulin variable
`
`domain, wherein the method comprises the following steps:
`
`(a)
`
`10 nucleic acids each encoding a rabbit-derived
`least
`providing at
`complementarity determining region 3 (CDR3) amino acid sequence or an
`
`amino acid sequence further encompassing 1, 2, or 3 amino acids N-terminal
`
`and/or C-terminal of the rabbit—derived CDR3 amino acid sequence,
`
`(b)
`
`generating a population of 20 or more nucleic acids, each encoding at least
`
`one protein comprising at least one immunoglobulin variable domain having a
`
`rabbit CDR3 amino acid sequence of step (a) embedded in essentially
`
`human framework sequences, wherein the human framework sequences
`
`comprise a first human framework region (FR1), a second human framework
`
`region (FR2), a third human framework region (FR3), and a fourth human
`
`framework region (FR4),
`
`such that
`
`the FR1 and FR2 regions are interspaced by a complementarity
`
`determining region 1
`
`(CDR1),
`
`the FR2 and FR3 regions are interspaced by a
`
`complementarity determining region 2 (CDR2), and the FR3 and FR4 regions are
`
`interspaced by a rabbit-derived CDR3 amino acid sequence,
`
`wherein the nucleic acid sequences encoding the CDR1 and CDR2 amino acid
`
`sequences are diversified among the population of nucleic acids encoding at least
`
`one protein comprising at least one immunoglobulin variable domain,
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`

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`WO 2016/173719
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`PCT/EP2016/000701
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`wherein each nucleic acid sequence encoding a CDR1 or CDR2 amino acid
`
`sequence is independently based
`
`i)
`
`ii)
`
`on a nucleic acid sequence encoding a human CDR1 or CDR2, respectively,
`or
`
`on a nucleic acid sequence encoding a rabbit CDR1 or CDR2, respectively,
`
`wherein at least some of the nucleic acid sequences encoding a CDR1 or CDR2
`
`amino acid sequence have been modified to encode at least one amino acid
`
`present in rabbit CDR1 or CDR2 amino acid sequences, respectively,
`
`in case of
`
`human CDR1 or CDR2, respectively, or to encode at least one amino acid present
`
`in human CDR1 or CDR2 amino acid sequences, respectively,
`
`in case of rabbit
`
`CDR1 or CDR2, respectively,
`
`and wherein the human FR1, FR2, FR3 and FR4 regions are human framework
`
`regions selected to provide a scaffold conducive for rabbit CDR3 amino acid
`
`sequences,
`
`and wherein the nucleic acid sequences encoding the rabbit-derived CDR3 amino ,
`
`acid sequences or the amino acid sequence further encompassing 1, 2, or 3
`
`amino acids N-terminal and/or C-terminal of the rabbit-derived CDR3 amino acid
`
`sequence are diversified among the population of nucleic acids encoding at least
`
`one protein comprising at least one immunoglobulin variable domain,
`
`and wherein at least 10 of the nucleic acids of the population encode different
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`CDR3 amino acid sequences,
`
`with the proviso:
`
`—
`
`-
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`30
`
`that the two C-terminal amino acids of FR2 are optionally non-human, and
`
`that the two C-terminal amino acids of FR3 are optionally non—human.
`
`In one further preferred embodiment, at least 50% of the nucleic acids of the
`
`population encode different CDR3 amino acid sequences.
`
`35
`
`The methods and populations allow for efficient mass humanization of rabbit
`
`antibodies. The rabbit
`
`is a species which is
`
`in particular suitable for mass
`
`humanization of antibodies raised in a non-human mammal for several reasons:
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`WO 2016/173719
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`Firstly, the majority of the rabbit repertoire can be mapped to two heavy chain
`
`frameworks and two light chain frameworks. By comparison, the human repertoire
`
`uses 50 heavy chain and 70 light chain frameworks, and the mouse repertoire
`
`uses over 100 heavy chain frameworks. Having only two frameworks greatly
`
`simplifies the “landscape of all possible humanizations", as it becomes possible to
`
`map all possible humanizations to two human V-gene heavy chain scaffolds and
`
`two V-gene light chain scaffolds. This reduces the cost and complexity of the
`
`library construction, and moreover,
`humanization.
`
`improves the chance of successful mass
`
`As an example, a hypothetical non-human antibody that requires a specific heavy
`
`and light chain scaffold to successfully humanize is to be considered.
`
`If a skilled
`
`person had to try all human heavy chain and light chain scaffold combinations,
`only one out of every 3500 possible heavy and light combinations (50 VH * 70 VL)
`would be capable of potentially accepting the graft. Consequently, 99.97% of the
`
`library would be useless. In contrast, with the rabbit where there are only 2 heavy
`
`chain scaffolds and 2 light chain scaffolds, one out of four combinations would be
`
`correct (2 VH * 2 VL), allowing 25% of the library to be a potential successful graft
`
`space for every clone. When considering a mass humanization of ~1000 lineages
`
`after an immunization, only the latter can mathematically succeed.
`
`Secondly, the rabbit produces high affinity antibodies using both a hyperdiverse
`
`CDR—H3 as well as a hyperdiverse CDR-L3. This is in contrast to mice and
`
`humans, where almost all of the diversity is driven by the CDR-H3. The rabbit thus
`
`has greater capacity to generate unique binders across a greater surface area of
`
`CDR3 loops. By effectively doubling the "specificity space" that is transferred by
`
`the method of the invention, this results in a higher probability of success during
`
`the mass humanization process.
`
`Thirdly, the rabbit undergoes gene conversion as an affinity maturation strategy.
`
`This process
`
`introduces abrupt changes in
`
`the frameworks not unlike a
`
`humanization: affinity matured binders are those clones that both resemble the
`
`initial scaffold frameworks and can tolerate this process. Thus, gene conversion
`
`likely selects for clones that are CDR-H3/-L3 driven in their specificity and can
`
`accommodate affinity maturation replacement in the scaffold CDR-H1/-H2/-L1/—L2
`
`regions, making them particularly well suited for humanization.
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`“A population of nucleic acids" is understood as 2 or more nucleic acids, preferably
`
`2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100, 150, 200 or more nucleic acids, wherein at least 2
`
`of the nucleic acids of the population exhibit different nucleic sequences,
`
`more preferably wherein at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100, 150, 200 or
`
`more nucleic acids of the population exhibit different nucleic sequences, and/or
`
`wherein at least 50%, at least 80%, at least 90%, at least 95%, at least 96%, at
`
`least 97%, at
`
`least 98%, at
`
`least 99% or 100% of the nucleic acids of the
`
`population exhibit different nucleic sequences.
`
`According to the method of the invention, an encoded protein comprises at least
`
`one immunoglobulin variable domain, preferably 1, 2, 3, 4 or more immunoglobulin
`variable domains, more preferably 1 or 2 immunoglobulin variable domains. For
`
`example, a variable heavy immunoglobulin domain may be paired with a variable
`
`light domain to provide an antigen binding site; such as in a scFv as described in
`
`the examples. Alternatively, independent regions, e.g., a variable heavy domain
`
`alone or a variable light domain alone may be used. An immunoglobulin variable
`
`domain comprises CDR1, CDR2 and CDR3 sequences.
`
`In particular, an
`
`immunoglobulin variable heavy domain comprises CDR-1H, CDR-2H and CDR-3H
`
`sequences, and an immunoglobulin variable light domain comprises CDR—1L,
`
`CDR-2L and CDR-3L sequences.
`
`Accordingly,
`
`in one preferred embodiment, the proteins of the population each
`
`comprise one (1) immunoglobulin variable domain having a rabbit—derived CDR3
`
`amino acid sequence embedded in essentially human framework sequences of
`
`the invention as described above. Preferably,
`
`the proteins comprising one
`
`immunoglobulin variable domain having a rabbit-derived CDR3 amino acid
`
`sequence embedded in essentially human framework sequences each comprise a
`
`VH domain, or a VL domain, or a heavy chain of an antibody or a fragment thereof
`
`comprising the VH domain, or a light chain of an antibody or a fragment thereof
`
`comprising the VL domain and/or is selected from a single domain antibody.
`
`In a
`
`more preferred embodiment,
`
`the population of proteins each comprising one
`
`immunoglobulin variable comprises at least one protein comprising a VH domain
`
`and comprises at least one protein comprising a VL domain. This allows for pairing
`
`within the population of proteins. Alternatively, the population may be paired with
`
`proteins of a separate population comprising a VH domain or VL domain
`
`respectively.
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`In another preferred embodiment, the proteins of the population each comprise 2,
`
`3, 4 or more immunoglobulin variable domains having a rabbit-derived CDR3
`
`amino acid sequence embedded in essentially human framework sequences of
`
`the invention as described above. In a more preferred embodiment, the proteins of
`
`the population each comprise 2 immunoglobulin variable domains having a rabbit—
`
`derived CDR3 amino acid sequence embedded in essentially human framework
`
`sequences of the invention as described above. It is preferred that the proteins of
`
`the population comprise a VH domain and a VL domain, or a heavy chain of an -
`
`antibody or a fragment thereof comprising the VH domain, and a light chain of an
`
`antibody or a fragment thereof comprising the VL domain or an scFv, even more
`
`preferably an scFv. An scFv library of the invention is described in the Examples.
`
`In further more preferred embodiment, the encoded proteins or proteins of the
`
`inventions are selected from an immunoglobulin molecule, a disulfide linked Fv, a
`
`monoclonal antibody, an scFv, a diabody, a multispecific antibody, a Fab, a Fab', a
`
`bispecific antibody; a F(ab')2, an scFv or an Fv, more preferably an scFv. An scFv
`
`library of the invention is described in the Examples.
`
`Further, in one preferred embodiment, the nucleic acids of the population encode
`
`proteins each comprising one immunoglobulin variable domain having a rabbit-
`
`derived CDR3 amino acid sequence embedded in essentially human framework
`
`sequences of the invention as described above. Preferably, the encoded proteins
`
`comprising one immunoglobulin variable domain having a rabbit-derived CDR3
`
`amino acid sequence embedded in essentially human framework sequences each
`
`comprise a VH domain, or a VL domain, or a heavy chain of an antibody or a
`
`fragment thereof comprising the VH domain, or a light chain of an antibody or a
`
`fragment thereof comprising the VL domain and/or are selected from a single
`
`domain antibody. In a more preferred embodiment, the population of nucleic acids
`
`encoding proteins each comprising one immunoglobulin variable comprises at
`
`least one nucleic acid encoding a protein comprising a VH domain and comprises
`
`at least one nucleic acid encoding a protein comprising a VL domain. This allows
`
`for pairing within the population of proteins encoded by the nucleic acids of the
`
`population. Alternatively,
`
`the population encoding proteins comprising one VLv
`
`domain or one VH domain only, may be paired with a separate population of
`
`nucleic acids encoding proteins comprising a VH domain or VL domain,
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`respectively.
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`In another preferred embodiment,
`
`the nucleic acids of the population encode
`
`proteins each comprising 2, 3, 4 or more immunoglobulin variable domains having
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`WO 2016/173719
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`PCT/EP2016/000701
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`a rabbit-derived CDR3 amino acid sequence embedded in essentially human
`
`framework sequences of the invention as described above.
`
`In a more preferred
`
`embodiment, the nucleic acids of the population encode proteins, wherein each
`
`protein comprises 2 immunoglobulin variable domains having a rabbit—derived
`
`CDR3 amino acid sequence embedded in
`
`essentially human framework
`
`sequences of the invention as described above.
`
`It is preferred that the encoded
`
`proteins of the population comprise a VH domain and a VL domain, or a heavy
`
`chain of an antibody or a fragment thereof comprising the VH domain, and a light
`
`chain of an antibody or a fragment thereof comprising the VL domain, or an scFv,
`
`even more preferably an scFv. An scFv library of the invention is described in the
`
`Examples.
`
`In further more preferred embodiment, the encoded proteins or proteins of the
`
`subject-matter of the invention are selected from an immunoglobulin molecule, a
`
`disulfide linked Fv, a monoclonal antibody, an scFv, a diabody, a multispecific
`
`antibody, a Fab, a Fab', a bispecific antibody; a F(ab')2, an scFv or an Fv, more
`
`preferably an scFv. An scFv library of the invention is described in the Examples.
`
`A “rabbit CDR3 amino acid sequence” is understood as an amino acid sequence
`
`which is identical
`
`to a CDR3 amino sequence naturally occurring in a rabbit
`
`antibody. CDR3 regions resulting after an immunization are also considered to be
`
`natural. The CDR3 amino sequence may be a CDR-3L or a CDR-3H amino acid
`
`sequence.
`
`A "rabbit—derived CDR3 amino acid sequence” is understood as an amino acid
`
`sequence which is identical to a CDR3 amino sequence naturally occurring in a
`
`rabbit antibody, or which contains 1, 2, 3, 4, or 5 amino acid mutations compared
`
`to a CDR3 amino sequence naturally occurring in a rabbit antibody, preferably
`
`wherein the mutation is a conservatiVe mutation.
`
`Conservative amino acid substitutions, as one of ordinary skill
`
`in the art will
`
`appreciate, are substitutions that replace an amino acid residue with one imparting
`
`similar or better
`
`(for
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`the
`
`intended purpose)
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`functional
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`and/or
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`chemical
`
`characteristics. For example, conservative amino acid substitutions are often ones
`
`in which the amino acid residue is replaced with an amino acid residue having a
`
`similar side chain. Families of amino acid residues having similar side chains have
`
`been defined in the art. These families include amino acids with basic side chains
`
`(e.g.,
`
`lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic
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`acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine,
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`threonine,
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`tyrosine, cysteine,
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`tryptophan), nonpolar side chains (e.g., alanine,
`
`valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side
`
`chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine,
`
`phenylalanine, tryptophan, histidine). The purpose for making a substitution is not
`
`significant and can include, but is by no means limited to, replacing a residue with
`
`one better able to maintain or enhance the structure of the molecule, the charge or
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`hydrophobicity of the molecule, or the size of the molecule. For instanCe, one may
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`desire simply to substitute a less desired residue with one of the same polarity or
`
`charge. Such modifications can be introduced by standard techniques known in
`
`the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. In the
`
`context of the present
`invention, a “conservative amino acid substitution” is
`preferably defined by a substitution within a class of amino acids reflected in the
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`following table:
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`Imto acid residue classes for conservative
`Amino acids
`
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`substitutions
`
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`Acidic residues (i.e. residues with acidic side
`Asp, Glu
`
`
`chain)
`Lys, Arg, His
`Basic residues (i.e. residues with basic side
`
`
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`L chain)
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`glycine, asparagine, glutamine,
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`
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`serine, threonine, tyrosine,
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`c steine, tr ootohan
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`alanine, valine, leucine,
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`
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`Nucleic acid molecules of the present invention may be in the form of RNA, such
`
`including, for instance, cDNA and
`as mRNA or cRNA, or in the form of DNA,
`genomic DNA e.g. obtained by cloning or produced by chemical synthetic
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`20
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`techniques or by a combination thereof. The DNA may be triple-stranded, double-
`
`stranded or single—stranded. Single—stranded DNA may be the coding strand, also
`
`known as the sense strand, or it may be the non-coding strand, also referred to as
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`Polar uncharged residues (i.e. residues with
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`uncharged polar side chain)
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`
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`Nonpolar Uncharged residues (i.e. residues
`
`
`
`
`with uncharged nonpolar side chain)
`isoleucine, proline,
`
`
` phen lalanine, methionine
`
`
`
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`threonine, valine, isoleucine
`Beta—branched residues (i.e. side chains with
`beta-branched side chain
`
`
`
`tyrosine, phenylalanine,
`Aromatic residues (i.e. residues with aromatic
`tr oto o han
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`the anti-sense strand. Nucleic acid molecule as used herein also refers to, among
`
`other, single- and double— stranded DNA, DNA that is a mixture of single— and
`
`double-stranded RNA, and RNA that is a mixture of single- and double-stranded
`
`regions, hybrid molecules comprising DNA and RNA that may be single-stranded
`
`or, more typically, double-stranded, or triple-stranded, or a mixture of single- and
`
`double-stranded regions. In addition, nucleic acid molecule as used herein refers
`
`to triple-stranded regions comprising RNA or DNA or both RNA and DNA.
`
`The nucleic acids may be originally formed in vitro or in a cell in culture, in general,
`
`by the manipulation of nucleic acids by endonucleases and/or exonucleases
`
`and/or polymerases and/or ligases and/or recombinases or other methods known
`
`to the skilled practitioner to produce the nucleic acids.
`
`The term “embedded in essentially human framework sequences” is understood
`
`as that the CDR3-derived sequence is located within the framework sequences to
`
`yield an immunoglobulin variable domain. For example, a skilled person is aware
`
`that a CDR-3L amino acid sequence is located between FR3 and FR4 framework
`
`regions of the light chain in case of an immunoglobulin light chain variable domain.
`
`“Human framework sequences” are understood as framework sequences which
`
`are naturally occurring human framework sequences. The nucleic acids encoding
`
`the human framework sequences may contain silent mutations as compared to the
`
`naturally occurring nucleic acids encoding the human framework sequences
`
`and/or sequences that are a result of the degeneration of the genetic code. There
`
`are 20 natural amino acids, most of which are specified by more than one codon.
`
`Therefore, all nucleotide sequences are included which result
`
`in the human
`
`framework sequences as defined above.
`
`An “essentially human framework sequence” is understood as a framework
`
`sequence which exhibits at least 90%, preferably at least 95%, 96%, 97%, 98%, or
`
`99% sequence identity to a naturally occurring human framework sequence. In a
`
`preferred embodiment,
`
`the essentially human framework sequence consists of
`
`FR‘I, FR2, FR3 and FR4 regions, which are human FR1, FR2, FR3 and FR4
`
`regions, with the proviso that the two C-terminal amino acids of FR2 are optionally
`
`non-human, and that the two C-terminal amino acids of FR3 are optionally non-
`
`human, more preferably,
`
`the two C-terminal amino acids of heavy FR2 are
`
`optionally non-human, and that the two C-terminal amino acids of heavy FR3 are
`
`optionally non-human.
`
`In an even more preferred embodiment, the non-human
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`amino acids of FR2 and/or FR3 are rabbit FR2 and/or FR3 amino acids of the
`
`corresponding positions.
`
`The percentage of sequence identity can be determined e.g. by sequence
`
`alignment. Methods of alignment of sequences for comparison are well known in
`
`the art. Various programs and alignment algorithms have been described e.g. in
`
`Smith and Waterman, Adv. Appl. Math. 2: 482, 1981 or Pearson and Lipman,
`
`Proc. Natl. Acad. Sci.US. A. 85: 2444, 1988.
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`The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol.
`
`215: 403-410, 1990) is available from several sources,
`
`including the National
`
`Center for Biotechnology Information (NCBI, Bethesda, MD) and on the Internet,
`
`for use in connection with the sequence analysis programs blastp, blastn, blastx,
`
`tblastn and tblastx. Amino acid sequences are typically characterized using the
`
`NCBI Blast 2.0, gapped blastp set to default parameters. For comparisons of
`
`amino acid sequences of at least 30 amino acids, the Blast 2 sequences function
`
`is employed using the default BLOSUM62 matrix set to default parameters, (gap
`
`existence cost of 11, and a per residue gap cost of 1). When aligning short
`
`peptides (fewer than around 30 amino acids), the alignment is performed using the
`Blast 2 sequences function, employing the PAM30 matrix set t default parameters
`
`20
`
`(open gap 9, extension gap 1 penalties). Methods for determining sequence
`
`identity over such short windows such as 15 amino acids or less are described at
`
`the website that
`
`is maintained by the National Center
`
`for Biotechnology
`
`Information in Bethesda, Maryland.
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`The percentage of sequence homology can be determined by counting the
`
`positions with identical amino acids plus the positions with conservative amino
`
`acid substitutions from an alignment produced with the method described above.
`
`“An amino acid sequence further encompassing 1, 2, or 3 amino acids N—terminal
`
`and/or C-terminal of the rabbit-derived CDR3 amino acid sequence” is understood
`
`as that the rabbit-derived CDR3 amino acid sequence, preferably the rabbit-CDR3
`
`amino acid sequence further comprises 1, 2, or 3 amino acids N-terminal of the
`
`rabbit—derived CDR3 amino acid sequence, and/or 1, 2, or 3 amino acids C-
`
`terminal of
`
`the rabbit-derived CDR3 amino acid sequence.
`
`in a preferred
`
`embodiment, the rabbit-derived CDR3 amino acid sequence further encompassing
`
`1, 2, or 3 amino acids N-terminal and/or C-terminal of‘the rabbit—derived CDR3
`
`amino acid sequence is a rabbit—derived amino acid sequence comprising a rabbit-
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`derived CDR3 amino acid sequence or rabbit CDR3 amino acid sequence, more
`
`preferably a rabbit amino acid sequence comprising a rabbit CDR3 amino acid
`
`sequence.
`
`A “rabbit-derived amino acid sequence“ is understood as an amino acid sequence
`
`which is identical to a amino sequence naturally according in a rabbit antibody, or
`
`which contains 1, 2, 3, 4, or 5 amino acid mutations compared to an amino
`
`sequence naturally occurring in a rabbit antibody, preferably wherein the mutation
`is a conservative mutation.
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`1O
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`In a preferred embodiment, the rabbit specificity