1-24. (canceled)
25. A circuit arrangement comprising:
an antenna port;
a transmission port;
a reception port;
three 90\xb0 hybrids, each 90\xb0 hybrid dividing an input signal into two output signals that have a relative phase shift of 90\xb0 with respect to one another, wherein the antenna port, the transmission port and the reception port are each connected to at least one 90\xb0 hybrid; and
two duplexers interconnected in such a way that the two output signals output by the 90\xb0 hybrid connected to the transmission port constructively interfere at the antenna port and parasitic signals caused by the two output signals destructively interfere at the reception port.
26. The circuit arrangement according to claim 25, wherein each duplexer includes three connections and wherein both duplexers have the three connections connected to a 90\xb0 hybrid.
27. The circuit arrangement according to claim 25,
wherein one of the two duplexers is connected to the 90\xb0 hybrid connected to the reception port and the 90\xb0 hybrid connected to the transmission port in such a way that the 90\xb0 hybrids each output an output signal, which is phase-shifted relative to its input signal through an angle \u03a61, to the duplexer;
wherein the other of the two duplexers is connected to the 90\xb0 hybrid connected to the reception port and the 90\xb0 hybrid connected to the transmission port in such a way that the 90\xb0 hybrids each output an output signal, which is phase-shifted relative to its input signal through an angle \u03a62, to the duplexer;
wherein a magnitude of the difference between the two angles \u03a61 and \u03a62 is approximately 90\xb0; and
wherein the two duplexers are each connected to one of the outputs of the 90\xb0 hybrid connected to the antenna port.
28. The circuit arrangement according to claim 27, wherein the angle \u03a61 is approximately equal to 0\xb0 and wherein the angle \u03a62 is approximately equal to 90\xb0.
29. The circuit arrangement according to claim 27, wherein the angle \u03a61 is approximately equal to \u221245\xb0 and wherein the angle \u03a62 is approximately equal to 45\xb0.
30. The circuit arrangement according to claim 25, wherein the two duplexers are each designed to be tunable in terms of frequency within a frequency band.
31. The circuit arrangement according to claim 30, wherein the duplexers are adjusted in such a way that an insertion loss of a transmission passband for the frequency of a transmission channel used at that time is minimal and increases considerably between the transmission channel used at that time and a reception channel used at that time.
32. The circuit arrangement according to claim 30, wherein the duplexers are adjusted such that an insertion loss of a reception passband for the frequency of a reception channel used at that time is minimal and increases considerably between a transmission channel used at that time and the reception channel used at that time.
33. The circuit arrangement according to claim 25,
wherein the 90\xb0 hybrids are formed from discrete elements, or
wherein the 90\xb0 hybrids are microstrip conductors.
34. The circuit arrangement according to claim 25,
wherein the duplexers are formed from discrete elements, or
wherein the duplexers contain acoustic components.
35. The circuit arrangement according to claim 34, in which the duplexers contain SAW duplexers, BAW duplexers or hybrid duplexers, which have both SAW transducers and BAW transducers.
36. The circuit arrangement according to claim 25, wherein the duplexers have high-pass filters and low-pass filters.
37. The circuit arrangement according to claim 25, wherein the duplexers have tunable elements.
38. The circuit arrangement according to claim 25, wherein the relative phase shifts of the 90\xb0 hybrids are adjusted such that asymmetries in a layout of the circuit arrangement are compensated for.
39. The circuit arrangement according to claim 25, wherein discrepancies in the relative phase shift of the output signals output by the 90\xb0 hybrids are compensated for by an asymmetric layout of the circuit arrangement.
40. The circuit arrangement according to claim 25,
wherein the circuit arrangement is designed for a plurality of different frequency bands;
wherein the circuit arrangement comprises two duplexers for each frequency band; and
wherein the circuit arrangement comprises a switchover circuit to switch between different duplexers and frequency bands.
41. The circuit arrangement according to claim 40, wherein the circuit arrangement comprises a separate reception port for each frequency band, each of the reception ports being connected to a dedicated 90\xb0 hybrid that includes outputs are interconnected with the two duplexers of the respective frequency band, wherein the switchover circuit is configured to connect the transmission port selectively to duplexers associated with different frequency bands.
42. The circuit arrangement according to claim 25, wherein the reception port is balanced.
43. The circuit arrangement according to claim 25, wherein the circuit arrangement has a diplexer.
44. A module, comprising a circuit arrangement according to claim 25.
45. The module according to claim 44, wherein duplexers andor the 90\xb0 hybrids are integrated in a substrate of the module.
46. The module according to claim 45, wherein the duplexers and the 90\xb0 hybrids are integrated in the form of L, C and R elements in a multilayered module substrate.
47. A device for wireless communication in the radio frequency range, the device comprising a front-end module according to claim 44.
48. A device for wireless communication in the radio frequency range, the device comprising a circuit arrangement according to claim 25.
49. A circuit arrangement comprising:
an antenna port;
a transmission port;
a reception port;
a first 90\xb0 hybrids with an input coupled to the reception port;
a first duplexer with an input coupled to a first output of the first 90\xb0 hybrid;
a second duplexer with an input coupled to a second output of the first 90\xb0 hybrid;
a second 90\xb0 hybrid with an input coupled to a first output of the first duplexer, a first output coupled to the antenna port and a second output coupled to a second output of the second duplexer; and
a third 90\xb0 hybrid with an input coupled to a first output of the second duplexer, a first output coupled to the transmission port and a second output coupled to a second output of the first duplexer.
The claims below are in addition to those above.
All refrences to claim(s) which appear below refer to the numbering after this setence.
1-97. (canceled)
98. A method of affinity maturation of a first antibody, or antigen-binding portion thereof, for a target antigen, comprising:
a) identifying a related antibody, or antigen-binding portion thereof, that exhibits a reduced activity for the target antigen than the corresponding form of a first antibody, wherein the related antibody, or antigen-binding portion thereof, contains a related variable heavy chain or a related variable light chain that is either:
one in which the corresponding variable heavy chain or variable light chain of the related antibody, or antigen-binding portion thereof, exhibits at least 75% amino acid sequence identity to the variable heavy chain or variable light chain of the first antibody, or antigen-binding portion thereof, but does not exhibit 100% sequence identity therewith; or
one in which at least one of the VH, DH, and JH germline segments of a nucleic acid molecule encoding the variable heavy chain of the related antibody, or antigen-binding portion thereof, is identical to one of the VH, DH, and JH germline segments of the nucleic acid molecule encoding the variable heavy chain of the first antibody, or antigen-binding portion thereof, andor at least one of the V\u03ba and J\u03ba or at least one of the V\u03bb and J\u03bb germline segments of the nucleic acid molecule encoding the variable light chain is identical to one of the V\u03ba and J\u03ba or V\u03bb and J\u03bb germline segments of the nucleic acid molecule encoding the variable light chain of the first antibody, or antigen-binding portion thereof; and
b) comparing the amino acid sequence of the variable heavy chain or variable light chain of the first antibody, or antigen-binding portion thereof, to the amino acid sequence of the corresponding related variable heavy chain or variable light chain of the related antibody, or antigen-binding portion thereof;
c) identifying a target region within the variable heavy chain or variable light chain of a first antibody, or antigen-binding portion thereof, wherein the target region exhibits at least one amino acid difference compared to the same region in the related antibody, or antigen-binding portion thereof;
d) producing a plurality of modified antibodies, or antigen-binding portions thereof, each comprising a variable heavy chain and a variable light chain, or a portion thereof, wherein at least one of the variable heavy chain or variable light chain is modified in its target region by replacement of a single amino acid residue, whereby the target region in each of the plurality of antibodies, or antigen-binding portions thereof, contains replacement of an amino acid to a different amino acid compared to the first antibody, or antigen-binding portion thereof;
e) screening each of the plurality of modified antibodies, or antigen-binding portions thereof, for an activity to the target antigen; and
f) selecting those modified antibodies, or antigen-binding portions thereof, that exhibit increased activity for the target antigen compared to the first antibody, or antigen-binding portion thereof.
99. A method according to claim 98 that further includes at least one of the following:
a) wherein the plurality of modified antibodies, or antigen-binding portions thereof, in part (d) are produced by producing a plurality of nucleic acid molecules that encode modified forms of a variable heavy chain or a variable light chain of the first antibody, or antigen-binding portion thereof, wherein the nucleic acid molecules contain one codon encoding an amino acid in the target region that encodes a different amino acid as compared to the unmodified variable heavy or variable light chain, whereby each nucleic acid molecule of the plurality encodes a variable heavy chain or variable light chain that is modified in its target region by replacement of a single amino acid residue;
b) wherein the target region in the first antibody, or antigen-binding portion thereof, exhibits 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid differences compared to the corresponding region in the related antibody, or antigen-binding portion thereof;
c) wherein the related antibody, or antigen-binding portion thereof, is 1, 2, 3, 4, or 5 related antibodies, or antigen-binding portions thereof;
d) wherein the activity is selected from the group consisting of:
i. binding, optionally binding as assessed by a method selected from the group consisting of an immunoassay, optionally an immunoassay selected from the group consisting of a radioimmunoassay, an enzyme linked immunosorbent assay (ELISA), and an electrochemiluminescence assay, wherein the electrochemiluminescence assay optionally is meso-scale discovery (MSD); whole cell panning; and surface plasmon resonance (SPR);
ii. signal transduction;
iii. differentiation;
iv. alteration of gene expression;
v. cellular proliferation;
vi. apoptosis;
vii. chemotaxis;
viii. cytotoxicity;
ix. cancer cell invasion;
x. endothelial cell proliferation; and
xi. tube formation;
e) wherein the first antibody, or antigen-binding portion thereof, binds to the target antigen when in a Fab form with a binding affinity that is about 10\u22124 M or lower, about 10\u22124 M to about 10\u22128 M, or at or about 10\u22124 M, 10\u22125 M, 10\u22126 M, 10\u22127 M, 10\u22128 M, or lower;
f) wherein the related antibody, or antigen-binding portion thereof, exhibits a binding affinity that is less than the binding affinity of the first antibody, or antigen-binding portion thereof, whereby the binding affinity of the related antibody, or antigen-binding portion thereof, in its Fab form is about 10\u22124 M or lower; about 10\u22124 M to about 10\u22128 M; or at or about 10\u22124 M, 10\u22125 M, 10\u22126 M, 10\u22127 M, 10\u22128 M, or lower;
g) wherein the related antibody, or antigen-binding portion thereof, exhibits about 80% or less activity than the corresponding form of the first antibody, or antigen-binding portion thereof; about 5% to about 80% of the activity of the corresponding form of the first antibody; or less than or about 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, or less activity than the corresponding form of the first antibody.
h) wherein the related antibody, or antigen-binding portion thereof, exhibits the same or similar level of activity to the target antigen compared to a negative control;
i) wherein the target region is identified within the variable heavy chain of the first antibody, or antigen-binding portion thereof, and steps d)-f) are performed therefrom;
j) wherein the target region is identified within the variable light chain of the first antibody, or antigen-binding portion thereof, and steps d)-f) are performed therefrom;
k) wherein:
a target region is identified within the variable heavy chain of the first antibody, or antigen-binding portion thereof, and steps d)-f) are performed therefrom; and
separately and independently a target region is identified within the variable light chain of the first antibody, or antigen-binding portion thereof, and steps d)-f) are performed therefrom;
l) wherein a related antibody, or antigen-binding portion thereof, that contains the related corresponding variable heavy chain is different than a related antibody, or antigen-binding portion thereof, that contains the related corresponding variable light chain;
m) wherein a related antibody, or antigen-binding portion thereof, that contains the related corresponding variable heavy chain is the same as a related antibody, or antigen-binding portion thereof, that contains the related corresponding variable light chain;
n) wherein the amino acid sequence of the variable heavy chain or variable light chain of the first antibody, or antigen-binding portion thereof, exhibits at least about 80% or more sequence identity with the corresponding amino acid sequence of the related variable heavy chain or variable light chain of the related antibody, or antigen-binding portion thereof; about 80% to about 99% of the sequence identity with the corresponding amino acid sequence of the related variable heavy chain or variable light chain of the related antibody, or antigen-binding portion thereof; or at least or about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the corresponding amino acid sequence of the related variable heavy chain or variable light chain of the related antibody, or antigen-binding portion thereof;
o) wherein the variable heavy chain or variable light chain of the first antibody, or antigen-binding portion thereof, exhibits at least about 95% sequence identity with the corresponding amino acid sequence of the related variable heavy chain or variable light chain of the related antibody, or antigen-binding portion thereof;
p) wherein the related antibody, or antigen-binding portion thereof, contains a related variable heavy chain or variable light chain that is one in which at least one of the VH, DH, and JH germline segments of the nucleic acid molecule encoding the variable heavy chain of the first antibody, or antigen-binding portion thereof, is identical to one of the VH, DH, and JH germline segments of the nucleic acid molecule encoding the variable heavy chain of the related antibody, or antigen-binding portion thereof; andor at least one of the V\u03ba and J\u03ba or at least one of the V\u03bb and J\u03bb germline segments of the nucleic acid molecule encoding the variable light chain of the first antibody, or antigen-binding portion thereof, is identical to one of the V\u03ba and J\u03ba or V\u03bb and J\u03bb germline segments of the nucleic acid molecule encoding the variable light chain of the related antibody, or antigen-binding portion thereof;
q) wherein the target antigen is selected from the group consisting of a polypeptide, carbohydrate, lipid, nucleic acid, and a small molecule;
r) wherein the target antigen is expressed on the surface of a virus, bacteria, tumor or other cell, or is a recombinant protein or peptide;
s) wherein the target antigen is a protein that is a target for therapeutic intervention, optionally selected from the group consisting of VEGFR-1, VEGFR-2, VEGFR-3 (vascular endothelial growth factor receptors 1, 2, and 3), a epidermal growth factor receptor (EGFR), ErbB-2, ErbB-3, IGF-R1, C-Met (also known as hepatocyte growth factor receptor; HGFR), DLL4, DDR1 (discoidin domain receptor), KIT (receptor for c-kit), FGFR1, FGFR2, FGFR4 (fibroblast growth factor receptors 1, 2, and 4), RON (recepteur d’origine nantais; also known as macrophage stimulating 1 receptor), TEK (endothelial-specific receptor tyrosine kinase), TIE (tyrosine kinase with immunoglobulin and epidermal growth factor homology domains receptor), CSF1R (colony stimulating factor 1 receptor), PDGFRB (platelet-derived growth factor receptor B), EPHA1, EPHA2, EPHB1 (erythropoietin-producing hepatocellular receptor A1, A2 and B1), TNF-R1, TNF-R2, HVEM, LT-\u03b2R, CD20, CD3, CD25, NOTCH, G-CSF-R, GM-CSF-R, EPO-R., a cadherin, an integrin, CD52, CD44, VEGF-A, VEGF-B, VEGF-C, VEGF-D, PIGF, EGF, HGF, TNF-\u03b1, LIGHT, BTLA, lymphotoxin (LT), IgE, G-CSF, GM-CSF and EPO;
t) wherein the target antigen is involved in cell proliferation and differentiation, cell migration, apoptosis or angiogenesis;
u) wherein a subset of the amino acid residues in the target region are modified by amino acid replacement;
v) wherein only the amino acid residues that differ between the first antibody and related antibody in the target region are modified by amino acid replacement;
w) wherein only the amino acid residues that are the same between the first antibody and the related antibody in the target region are modified by amino acid replacement;
x) wherein all of the amino acids residues in the target region are modified by amino acid replacement;
y) wherein each amino acid residue that is modified in the target region is modified to all 19 other amino acid residues, or a restricted subset thereof;
z) further comprising determining the amino acid modifications that are altered in the modified antibody compared to the first antibody not containing the amino acid replacements;
aa) wherein the method is repeated iteratively, wherein a modified antibody, or antigen-binding portion thereof, is selected and used in step a) as the first antibody, or antigen-binding portion thereof, for subsequent affinity maturation thereof;
bb) wherein one or more amino acid replacements in the target region of one or more variable heavy chains or one or more variable light chains of selected modified antibodies, or antigen-binding portions thereof, are combined to generate a further modified antibody, or antigen-binding portion thereof, whereby the further modified antibody(ies), or antigen-binding portion(s) thereof, are screened for an activity to the target antigen to identify a further modified antibody, or antigen-binding portion thereof, that exhibits an increased activity for the target antigen compared to the first antibody, or antigen-binding portion thereof, and to the selected modified antibody(ies), or antigen-binding portion(s) thereof; and
cc) wherein the antibody, or antigen-binding portion thereof, comprising a variable heavy chain and a variable light chain, or a portion thereof, is selected from the group consisting of a Fab, Fab\u2032, F(ab\u2032)2, single-chain Fv (scFv), scFab, Fv, dsFv, diabody, Fd, Fd\u2032, Fab fragment, Fd fragment, Fd\u2032 fragment, scFv fragment, and scFab fragment.
100. A method according to claim 98, wherein the related antibody, or antigen-binding portion thereof, contains a related variable heavy chain or variable light that is one in which at least one of the VH, DH, and JH germline segments of the nucleic acid molecule encoding the variable heavy chain of the first antibody, or antigen-binding portion thereof, is from the same gene family as one of the VH, DH, and JH germline segments of the nucleic acid molecule encoding the variable heavy chain of the related antibody, or antigen-binding portion thereof; andor at least one of the V\u03ba and J\u03ba or at least one of the V\u03bb and J\u03bb germline segments of the nucleic acid molecule encoding the variable light chain of the first antibody, or antigen-binding portion thereof, is from the same gene family as one of the V\u03ba and J\u03ba or V\u03bb and J\u03bb germline segments of the nucleic acid molecule encoding the variable light chain of the related antibody, or antigen-binding portion thereof.
101. A method according to claim 98, wherein the variable heavy chain or variable light chain of the first antibody, or antigen binding portion thereof, exhibits at least 60% or more sequence identity with the corresponding related variable heavy chain or variable light chain of the related antibody, or antigen binding portion thereof; 60% to 99% of the sequence identity with the corresponding related variable heavy chain or variable light chain of the related antibody, or antigen binding portion thereof; or at least or about 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the corresponding related variable heavy chain or variable light chain of the related antibody, or antigen binding portion thereof.
102. A method according to claim 98, wherein the target region is selected from the group consisting of a CDR1, CDR2, CDR3, FR1, FR2, FR3, and FR4.
103. A method according to claim 98, wherein:
a) the first antibody, or antigen-binding portion thereof, is identified by screening a combinatorial antibody library or combinatorial antigen-binding antibody fragment library;
b) the combinatorial library is produced by a method comprising:
i) combining a VH, a DH, and a JH human germline segment or portion thereof in frame to generate a sequence of a nucleic acid molecule encoding a VH chain or a portion thereof;
ii) combining a V\u03ba and a J\u03ba human germline segment or portion thereof, or a V\u03bb and a J\u03bb germline segment or portion thereof in frame to generate a sequence of a nucleic acid molecule encoding a VL chain or a portion thereof, wherein:
in steps i) and ii), each of the portions of the VH, DH, JH, V\u03ba, J\u03ba, V\u03bb, or J\u03bb are sufficient to produce an antibody or antigen-binding portion thereof containing a VH or VL or portion thereof that forms a sufficient antigen binding site;
iii) repeating steps i) and ii) a plurality of times to generate sequences of a plurality of different nucleic acid molecules;
iv) synthesizing the nucleic acid molecules to produce two libraries, wherein:
the first library comprises nucleic acid molecules encoding a VH chain or a portion thereof; and
the second library comprises nucleic acid molecules encoding a VL chain or a portion thereof;
v) introducing a nucleic acid molecule from the first library and from the second library into a cell and repeating this a plurality of times to produce a library of cells, wherein each cell contains nucleic acid molecules encoding a different combination of VH and VL from at least some of the other cells in the library of cells; and
vi) growing the cells to express the antibodies, or antigen-binding portions thereof, thereby producing a plurality of antibodies, or antigen-binding portion thereof, wherein the different antibodies, or antigen-binding portions thereof, in the library each comprise a different combination of a VH and a VL chain or a sufficient portion thereof to form an antigen binding site; and
c) screening of the library is effected by:
i) contacting an antibody, or antigen-binding portion thereof, in the library with a target protein;
ii) assessing binding of the antibody, or antigen-binding portion thereof, with the target protein andor whether the antibody, or antigen-binding portion thereof, modulates a functional activity of the target protein; and
iii) identifying an antibody, or antigen-binding portion thereof, that exhibits an activity for the target protein, wherein the identified antibody, or antigen-binding portion thereof, is a first antibody.
104. A method according to claim 103 that further includes at least one of the following:
a) the related antibody also is identified by screening a combinatorial antibody library by steps a)-c), whereby the related antibody exhibits reduced activity for the target antigen compared to the first antibody;
b) the library is an addressable library, whereby:
in step iv), the synthesized nucleic acid sequences are individually addressed, thereby generating a first addressed nucleic acid library and a second addressed nucleic acid library;
in step v), the cells are addressed, wherein each locus comprises a cell that contains nucleic acid molecules encoding a different combination of a VH and a VL from every other cell in the addressed library of cells; and
in step vi) the plurality of antibodies or portions thereof are addressed, wherein:
the antibodies or portions thereof at each locus in the library are the same antibody and are different from those at each and every other locus; and
the identity of the antibody or portion thereof is known by its address, wherein optionally the antibodies in the addressable library are arranged in a spatial array, optionally a multiwell plate, wherein each individual locus of the array corresponds to a different antibody member;
c) wherein the antibodies are in an addressable library, wherein optionally the antibodies in the addressable library are arranged in a spatial array, optionally a multiwell plate, wherein optionally each individual locus of the array corresponds to a different antibody member are attached to a solid support selected from the group consisting of a filter, chip, slide, bead or cellulose, and the different antibody members are immobilized to the surface thereof;
d) wherein the plurality of nucleic acid molecules are generated by a method selected from the group consisting of PCR mutagenesis, cassette mutagenesis, site-directed mutagenesis, random point mutagenesis, mutagenesis using uracil containing templates, oligonucleotide-directed mutagenesis, phosphorothioate-modified DNA mutagenesis, mutagenesis using gapped duplex DNA, point mismatch repair, mutagenesis using repair-deficient host strains, restriction-selection and restriction-purification, deletion mutagenesis, mutagenesis by total gene synthesis, and double-strand break repair; and
e) wherein the plurality of nucleic acid molecules are generated by a method selected from the group consisting of NNK, NNS, NNN, NNY or NNR mutagenesis.
105. A method according to claim 98, further comprising before step d),
g) performing scanning mutagenesis of the first antibody, or antigen-binding portion thereof, comprising producing a plurality of modified antibodies, or antigen-binding portions thereof, comprising a variable heavy chain and a variable light chain, or a portion thereof, wherein at least one of the variable heavy chain or variable light chain, or portion thereof, is one that is modified by replacement of a single amino acid residue with a scanned amino acid residue in the target region, whereby each of the plurality of antibodies, or antigen-binding portion thereof, contains replacement of an amino acid in the target region compared to the first antibody, or antigen-binding portion thereof, wherein the scanned amino acid optionally is selected from the group consisting of alanine, threonine, proline, glycine, and a non-natural amino acid;
h) screening each of the plurality of modified antibodies, or antigen-binding portions thereof, for an activity to the target antigen; and
i) selecting a second antibody, or antigen-binding portion thereof, from among the modified antibodies, or antigen-binding portions thereof, that exhibits retained or increased activity for the target antigen compared to the first antibody, or antigen-binding portion thereof, not containing the amino acid replacement, whereby the second antibody, or antigen-binding portion thereof, is used in place of the first antibody, or antigen-binding portion thereof, in step b).
106. A method according to claim 105 that further includes at least one of the following:
a) wherein the plurality of modified antibodies, or antigen-binding portions thereof, in step g) are produced by producing a plurality of nucleic acid molecules that encode modified forms of a variable heavy chain or a variable light chain of the first antibody, or antigen-binding portion thereof, containing the target region, wherein the nucleic acid molecules contain one codon that encodes a scanned amino acid in the target region compared to the corresponding codon of the unmodified variable heavy or variable light chain that does not encode the scanned amino acid, whereby each nucleic acid molecule of the plurality encodes a variable heavy chain or variable light chain that is modified by replacement of a single amino acid residue to the same scanned amino acid residue in the target region;
b) wherein a second antibody is, or antigen-binding portion thereof, selected that exhibits an activity that is at least 75% or more of the activity of the corresponding form of the first antibody, or antigen-binding portion thereof; is at least 75% to 200% of the activity of the corresponding form of the first antibody, or antigen-binding portion thereof; or is at least or about 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 130%, 140%, 150%, 200% or more of the activity of the corresponding form of the first antibody, or antigen-binding portion thereof;
c) further comprising after step i) determining the amino acid residue position that is modified in the second antibody, or antigen-binding portion thereof, to contain a neutral amino acid compared to the first antibody not containing the amino acid replacement;
d) wherein a subset of the amino acid residues in the target region are modified by amino acid replacement to a scanned amino acid;
e) wherein only the amino acid residues that differ between the first antibody, or antigen-binding portion thereof, and related antibody, or antigen-binding portion thereof, in the target region are modified by amino acid replacement to a scanned amino acid;
f) wherein all of the amino acids in the target region are modified by amino acid replacement to a scanned amino acid;
g) wherein the selected modified antibody, or antigen-binding portion thereof, exhibits about 2-fold, 5-fold, 10-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, 2000-fold, 3000-fold, 4000-fold, 5000-fold, 10000-fold, or more improved activity for the target antigen compared to the first antibody, or antigen-binding portion thereof; and
h) wherein the modified antibody, or antigen-binding portion thereof, exhibits a binding affinity that is greater than the binding affinity of the first antibody, or antigen-binding portion thereof, and is about 1\xd710\u22129 M or less; 1\xd710\u22129 M to 1\xd710\u221211 M; or is or is about 1\xd710\u22129 M, 2\xd710\u22129 M, 3\xd710\u22129 M, 4\xd710\u22129 M, 5\xd710\u22129 M, 6\xd710\u22129 M, 7\xd710\u22129 M, 8\xd710\u22129 M, 9\xd710\u22129 M, 1\xd710\u221210 M, 2\xd710\u221210 M, 3\xd710\u221210 M, 4\xd710\u221210 M, 5\xd710\u221210 M, 6\xd710\u221210 M, 7\xd710\u221210 M, 8\xd710\u221210 M, 9\xd710\u221210 M, or less
107. A method according to claim 98, comprising:
performing steps a)-f) on the variable heavy chain of the first antibody, or antigen-binding portion thereof, and selecting first modified antibodies, or antigen-binding portions thereof, each containing an amino acid replacement in the target region;
performing steps a)-f) independently and separately on the variable light chain of the first antibody and selecting second modified antibodies, or antigen-binding portions thereof, each containing an amino acid replacement in the target region;
combining the variable heavy chain of a first modified antibody, or antigen-binding portion thereof, with the variable light chain of a second modified antibody, or antigen-binding portion thereof, to generate a plurality of different third modified antibodies, or antigen-binding portions thereof, each comprising an amino acid replacement in the target region of the variable heavy chain and variable light chain; and
screening each of the plurality of third modified antibodies, or antigen-binding portions thereof, for binding to the target antigen; and
selecting those third modified antibodies, or antigen-binding portions thereof, that exhibit an increased activity for the target antigen compared to the first and second modified antibodies.
108. A method according to claim 98, further comprising after selecting a first modified antibody, or antigen-binding portion thereof, in step f):
j) selecting another different region within the variable heavy chain or variable light chain of the first modified antibody, or antigen-binding portion thereof, for further mutagenesis, wherein optionally the further different region is selected from the group consisting of a CDR1, CDR2, CDR3, FR1, FR2, FR3, and FR4;
k) producing a plurality of nucleic acid molecules that encode modified forms of the variable heavy chain or variable light chain of the first modified antibody, or antigen-binding portion thereof, wherein the nucleic acid molecules contain one codon encoding an amino acid in the selected region that encodes a different amino acid from the first modified variable heavy or variable light chain, whereby each nucleic acid molecule of the plurality encodes a variable heavy chain or variable light chain that is modified in the selected region by replacement of a single amino acid residue;
l) producing a plurality of further modified antibodies, or antigen-binding portions thereof, each comprising a variable heavy chain and a variable light chain, or a portion thereof, wherein at least one of the variable heavy chain or variable light chain is one produced in step k), whereby the selected region in each of the plurality of antibodies, or antigen-binding portions thereof, contains replacement of an amino acid to a different amino acid compared to the first modified antibody, or antigen-binding portion thereof;
m) screening each of the plurality of further modified antibodies, or antigen-binding portions thereof, for binding to the target antigen; and
n) selecting those further modified antibodies, or antigen-binding portions thereof, that exhibit increased activity for the target antigen compared to the first modified antibody, or antigen-binding portion thereof.
109. A method of affinity maturation of an antibody, or antigen-binding portion thereof, for a target antigen, comprising:
a) performing scanning mutagenesis of a first antibody, or antigen-binding portion thereof, comprising producing a plurality of nucleic acid molecules that encode modified forms of a variable heavy chain or a variable light chain of a first antibody, or antigen-binding portion thereof, wherein the nucleic acid molecules contain one codon that encodes another amino acid compared to the corresponding codon of the unmodified variable heavy or variable light chain, or portion thereof, that does not encode the other amino acid, whereby each nucleic acid molecule of the plurality encodes a variable heavy chain or variable light chain, or portion thereof, that is modified by replacement of a single amino acid residue to another amino acid such that every position across the full-length of the encoded variable heavy or light chain, or portion thereof, is replaced or every position in a selected region of the encoded variable heavy or variable light chain, or portion thereof, is replaced, whereby each replacement is to the same amino acid residue;
b) producing a plurality of modified antibodies, or antigen-binding portions thereof, each comprising a variable heavy chain and a variable light chain, or a portion thereof, wherein at least one of the variable heavy chain or variable light chain, or portion thereof, is one produced in step a), whereby each of the plurality of antibodies, or antigen-binding portions thereof, contains replacement of an amino acid position with another amino acid compared to the first antibody, or antigen-binding portion thereof;
c) screening each of the plurality of modified antibodies, or antigen-binding portions thereof, for an activity to the target antigen;
d) selecting a second antibody, or antigen-binding portion thereof, from among the modified antibodies, or antigen-binding portions thereof, that exhibits retained or increased activity for the target antigen compared to the first antibody, or antigen-binding portion thereof, not containing the amino acid replacement;
e) performing further mutagenesis of the second antibody, or antigen-binding portion thereof, comprising producing a plurality of nucleic acid molecules that encode modified forms of a variable heavy chain or a variable light chain, or portion thereof, of the second antibody, or antigen-binding portion thereof, wherein the nucleic acid molecules contain one codon encoding an amino acid at the scanned amino acid position that encodes a different amino acid than the scanned amino acid in the second antibody, or antigen-binding portion thereof, whereby each nucleic acid molecule of the plurality encodes a variable heavy chain or variable light chain, or portion thereof, that is modified at the scanned amino acid position by a single amino acid residue; and
f) producing a plurality of further modified antibodies, or antigen-binding portions thereof, each comprising a variable heavy chain and a variable light chain, or a portion thereof, wherein at least one of the variable heavy chain or variable light chain, or portion thereof, is one produced in step e), whereby the scanned amino acid position contains replacement to a different amino acid compared to the second antibody, or antigen-binding portion thereof;
g) screening each of the plurality of further modified antibodies, or antigen-binding portions thereof, for an activity to the target antigen; and
h) selecting a third antibody, or antigen-binding portion thereof, that exhibits increased activity for the target antigen compared to the first andor second antibody, or antigen-binding portion(s) thereof.
110. A method according to claim 109 that further includes at least one of the following:
a) wherein in step a) every position in a region of the encoded variable heavy or variable light chain is replaced;
b) wherein the selected region is a complementary determining region in the variable heavy chain or variable light chain selected from the group consisting of a CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3;
c) wherein a second antibody, or antigen-binding portion thereof, is selected that exhibits an activity that is at least 75% or more of the activity of the corresponding form of the first antibody; is 75% to 200% of the activity of the corresponding form of the first antibody, or antigen-binding portion thereof; or is at least or about 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 130%, 140%, 150%, 200% or more of the activity of the corresponding form of the first antibody, or antigen-binding portion thereof;
d) a method further comprising after step d) determining the amino acid residue position that is modified in the second antibody, or antigen-binding portion thereof, to contain a scanned amino acid compared to the first antibody, or antigen-binding portion thereof, not containing the amino acid replacement;
e) wherein the other amino acid is selected from the group consisting of alanine, threonine, proline, glycine, and non-natural amino acid;
f) wherein each of the plurality of nucleic acid molecules encodes a variable heavy chain or variable light chain, or portion thereof, that is modified by replacement of a single amino acid residue to the same scanned amino acid;
g) wherein an activity is selected from the group consisting of binding, signal transduction, differentiation, alteration of gene expression, cellular proliferation, apoptosis, chemotaxis, cytotoxicity, cancer cell invasion, endothelial cell proliferation and tube formation;
h) wherein the activity is selected from the group consisting of:
i. binding, optionally binding as assessed by a method selected from the group consisting of an immunoassay, optionally an immunoassay selected from the group consisting of a radioimmunoassay, an enzyme linked immunosorbent assay (ELISA), and an electrochemiluminescence assay, wherein the electrochemiluminescence assay optionally is meso-scale discovery (MSD); whole cell panning; and surface plasmon resonance (SPR);
ii. signal transduction;
iii. differentiation;
iv. alteration of gene expression;
v. cellular proliferation;
vi. apoptosis;
vii. chemotaxis;
viii. cytotoxicity;
ix. cancer cell invasion;
x. endothelial cell proliferation; and
xi. tube formation;
i) wherein the plurality of nucleic acid molecules produced in step e) are generated by a method selected from the group consisting of PCR mutagenesis, cassette mutagenesis, site-directed mutagenesis, random point mutagenesis, mutagenesis using uracil containing templates, oligonucleotide-directed mutagenesis, phosphorothioate-modified DNA mutagenesis, mutagenesis using gapped duplex DNA, point mismatch repair, mutagenesis using repair-deficient host strains, restriction-selection and restriction-purification, deletion mutagenesis, mutagenesis by total gene synthesis, and double-strand break repair;
j) wherein the plurality of nucleic acid molecules produced in step e) are generated by a method selected from the group consisting of NNK, NNS, NNN, NNY or NNR mutagenesis
k) wherein in step e), the scanned amino acid position is modified by amino acid replacement to all other amino acid residues, or to a restricted subset thereof, wherein optionally the modification does not include amino acid replacement to the scanned amino acid or to the original amino acid at that position in the first antibody, or antigen-binding portion thereof;
l) wherein the target antigen is selected from the group consisting of a polypeptide, carbohydrate, lipid, nucleic acid, and a small molecule;
m) wherein the target antigen is expressed on the surface of a virus, bacteria, tumor or other cell, or is a recombinant protein or peptide;
n) wherein the target antigen is a protein that is a target for therapeutic intervention;
o) wherein the target antigen is involved in cell proliferation and differentiation, cell migration, apoptosis or angiogenesis;
p) wherein the target antigen is selected from the group consisting of a VEGFR-1, VEGFR-2, VEGFR-3 (vascular endothelial growth factor receptors 1, 2, and 3), a epidermal growth factor receptor (EGFR), ErbB-2, ErbB-3, IGF-R1, C-Met (also known as hepatocyte growth factor receptor; HGFR), DLL4, DDR1 (discoidin domain receptor), KIT (receptor for c-kit), FGFR1, FGFR2, FGFR4 (fibroblast growth factor receptors 1, 2, and 4), RON (recepteur d’origine nantais; also known as macrophage stimulating 1 receptor), TEK (endothelial-specific receptor tyrosine kinase), TIE (tyrosine kinase with immunoglobulin and epidermal growth factor homology domains receptor), CSF1R (colony stimulating factor 1 receptor), PDGFRB (platelet-derived growth factor receptor B), EPHA1, EPHA2, EPHB1 (erythropoietin-producing hepatocellular receptor A1, A2 and B1), TNF-R1, TNF-R2, HVEM, LT-\u03b2R, CD20, CD3, CD25, NOTCH, G-CSF-R, GM-CSF-R, EPO-R., a cadherin, an integrin, CD52, CD44, VEGF-A, VEGF-B, VEGF-C, VEGF-D, PIGF, EGF, HGF, TNF-\u03b1, LIGHT, BTLA, lymphotoxin (LT), IgE, G-CSF, GM-CSF and EPO;
q) wherein the first antibody, or antigen-binding portion thereof, binds to the target antigen with a binding affinity when the antibody, or antigen-binding portion thereof, is in a Fab form that is about 10\u22124 M or lower; about 10\u22124 M to about 10\u22128 M; or that is at or about 10\u22124 M, 10\u22125 M, 10\u22126 M, 10\u22127 M, 10\u22128 M, or lower;
r) wherein scanning mutagenesis is performed within the variable heavy chain of the first antibody, or antigen-binding portion thereof, and steps a)-h) are performed therefrom;
s) wherein scanning mutagenesis is performed within the variable light chain of the first antibody, or antigen-binding portion thereof, and steps a)-h) are performed therefrom
t) wherein scanning mutagenesis is performed within the variable heavy chain of the first antibody and steps a)-h) are performed therefrom, and separately and independently, scanning mutagenesis is performed within the variable light chain of the first antibody, and steps a)-h) are performed therefrom;
u) wherein the third antibody, or antigen-binding portion thereof, exhibits at least 2-fold improved activity for the target antigen compared to the first andor second antibody(ies), or antigen-binding portion(s) thereof; 2-fold to 10000-fold or 2-fold to 1000-fold improved activity for the target antigen compared to the first andor second antibody(ies), or antigen-binding portion(s) thereof; or at least 2-fold, 5-fold, 10-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, 2000-fold, 3000-fold, 4000-fold, 5000-fold, 10000-fold or more improved activity for the target antigen compared to the first andor second antibody(ies), or antigen-binding portion(s) thereof;
v) wherein the third antibody, or antigen-binding portion thereof, exhibits a binding affinity that is greater than the binding affinity of the first antibody, or antigen-binding portion thereof, and is about 1\xd710\u22129 M or less; is 1\xd710\u22129 M to 1\xd710\u221211 M; or is or is about 1\xd710\u22129 M, 2\xd710\u22129 M, 3\xd710\u22129 M, 4\xd710\u22129 M, 5\xd710\u22129 M, 6\xd710\u22129 M, 7\xd710\u22129 M, 8\xd710\u22129 M, 9\xd710\u22129 M, 1\xd710\u221210 M, 2\xd710\u221210 M, 3\xd710\u221210 M, 4\xd710\u221210 M, 5\xd710\u221210 M, 6\xd710\u221210 M, 7\xd710\u221210 M, 8\xd710\u221210 M, 9\xd710\u221210 M, or less;
w) further comprising determining the amino acid modifications that are altered in the third antibody, or antigen-binding portion thereof, compared to the first antibody, or antigen-binding portion thereof, not containing the amino acid replacements;
x) wherein the method is repeated iteratively, and wherein the third antibody, or antigen-binding portion thereof, identified in step h) is selected and used in step a) as the first antibody, or antigen-binding portion thereof, for subsequent maturation thereof, whereby the amino acid residue that is modified is not further modified in subsequent iterations of the method;
y) wherein one or more amino acid replacement in one or more variable heavy chains or one or more variable light chains, or portions thereof, of selected third antibodies, or antigen-binding portions thereof, are combined to generate a further modified antibody, or antigen-binding portion thereof, whereby the further modified antibodies, or antigen-binding portions thereof, are screened for an activity to the target antigen to identify a further modified antibody, or antigen-binding portion thereof, that exhibits an increased activity for the target antigen compared to the first, second, andor third antibodies, or antigen-binding portions thereof; and
z) wherein the method comprises performing steps a)-h) on the variable heavy chain, or portion thereof, of the first antibody, or antigen-binding portion thereof, and selecting third antibodies, or antigen-binding portion thereof, each containing an amino acid replacement in the variable heavy chain, or portion thereof, compared to the corresponding variable heavy chain, or portion thereof, of the first antibody, or antigen-binding portion thereof;
performing steps a)-h) independently and separately on the variable light chain, or portion thereof, of the first antibody, or antigen-binding portion thereof, and selecting different third modified antibodies, or antigen-binding portions thereof, each containing an amino replacement in the variable light chain, or portion thereof, compared to the corresponding variable light chain, or portion thereof, of the first antibody;
combining the variable heavy chain, or portion thereof, of a third antibody, or antigen-binding portion thereof, with the variable light chain, or portion thereof, of a different third antibody, or antigen-binding portion thereof, to generate a plurality of different further modified antibodies, or antigen-binding portions thereof, each comprising an amino acid replacement of the variable heavy chain, or portion thereof, and variable light chain, or portion thereof, compared to the corresponding variable heavy or light chains, or portion(s) thereof, of the first antibody, or antigen-binding portion thereof;
screening each of the plurality of further modified antibodies, or antigen-binding portions thereof, for binding to the target antigen; and
selecting those fourth antibodies, or antigen-binding portions thereof, that exhibit an increased activity for the target antigen compared to the first, second, andor third antibodies, or antigen-binding portions thereof; and
aa) wherein the antibody, or antigen-binding portion thereof, comprising a variable heavy chain and a variable light chain, or a portion thereof, is selected from the group consisting of a Fab, Fab\u2032, F(ab\u2032)2, single-chain Fv (scFv), scFab, Fv, dsFv, diabody, Fd, Fd\u2032, Fab fragment, Fd fragment, Fd\u2032 fragment, scFv fragment, and scFab fragment.
111. A method according to claim 109, further comprising after selecting a third antibody in step h),
i) selecting another different region within the variable heavy chain or variable light chain of the third antibody, or antigen-binding portion thereof, optionally a different region selected from the group consisting of a CDR1, CDR2, CDR3, FR1, FR2, FR3, and FR4, for further mutagenesis;
j) producing a plurality of nucleic acid molecules that encode modified forms of the variable heavy chain or variable light chain of the third antibody, or antigen-binding portion thereof, wherein the nucleic acids molecules contain one codon encoding an amino acid in the selected region that encodes a different amino acid from the first modified variable heavy or variable light chain, whereby each nucleic acid molecule of the plurality encodes a variable heavy chain or variable light chain that is modified in the selected region by replacement of a single amino acid residue;
k) producing a plurality of further modified antibodies each comprising a variable heavy chain and a variable light chain, or a portion thereof, wherein at least one of the variable heavy chain or variable light chain is one produced in step j), whereby the selected region in each of the plurality of antibodies, or antigen-binding portions thereof, contains replacement of an amino acid to a different amino acid compared to the third antibody, or antigen-binding portion thereof;
l) screening each of the plurality of further modified antibodies, or antigen-binding portions thereof, for binding to the target antigen; and
m) selecting those further modified antibodies, or antigen-binding portions thereof, that exhibit increased activity for the target antigen compared to the third antibody, or antigen-binding portion thereof.