Monobodies selective to KRAS(G12D) with improved biophysical properties

Abstract

Disclosed are KRAS binding polypeptides that include a fibronectin type III domain having one or more modified loop sequences comprising a modified FG loop amino acid sequence, a modified BC loop amino acid sequence, a modified DE loop amino acid sequence, or a combination thereof, and optionally one or more modified beta strand sequences, wherein the one or more modified loop sequences and the one or more optionally modified beta strand sequences enable binding to a mutant KRAS having a G12D substitution at residue 12 (KRASG12D) and the binding polypeptide does not bind to wildtype KRASG12. Also disclosed are conjugates and fusion constructs containing the mutant KRAS binding polypeptide, polynucleotides encoding the KRAS binding polypeptides, fusion constructs and / or conjugates, delivery vehicles that contain the KRAS binding polypeptides, fusion constructs, conjugates, or polynucleotides, pharmaceutical compositions, as well as the diagnostic and therapeutic use of these materials.

Description

MONOBODIES SELECTIVE TO KRAS(G12D) WITH IMPROVED BIOPHYSICAL PROPERTIES

[0001] This application claims the priority benefit of U.S. Provisional Patent Application Serial No.63 / 507,889, filed June 13, 2023, which is hereby incorporated by reference in its entirety.

[0002] This invention was made with government support under grants R21 CA246457, R01 CA212608, and R01 CA194864 from the National Institutes of Health. The government has certain rights in the invention.

[0003] The Sequence Listing is being submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on May 14, 2024, is named 147462.002333.xml and is 34,673 bytes in size. No new matter is being introduced. FIELD OF THE INVENTION

[0004] The disclosed invention relates to novel KRAS binding polypeptides that selectively bind to a mutant KRAS having a G12D substitution at residue 12 of KRAS (KRASG12D) but exhibit insignificant binding to wildtype KRASG12. Also disclosed are conjugates and fusion constructs containing the novel KRAS binding polypeptides, as well as polynucleotides and delivery vehicles, and the use of these materials for treating KRAS- mediated cancers. BACKGROUND OF THE INVENTION

[0005] KRAS, part of the RAS / MAPK pathway, is a GTPase that converts GTP into GDP, and relays signals from outside the cell to the cell’s nucleus. To transmit signals to the nucleus, KRAS must be turned on by binding to GTP. When KRAS converts GTP to GDP, it becomes inactivated and does not relay signals to the nucleus. Thus, KRAS is turned on and off by the GTP and GDP molecules. During normal functioning, these signals instruct the cell to grow and proliferate or to mature and differentiate.

[0006] Activating, missense mutations of RAS genes (KRAS, HRAS and NRAS) are frequently associated with human cancers and play important roles in oncogenic transformation (Cox et al., “Drugging the Undruggable RAS: Mission Possible? Nat Rev Drug Discov 13:828- 851 (2014); Moore et al., “RAS-targeted Therapies: Is the Undruggable Drugged? Nat Rev Drug Discov 19:533-552 (2020)). Oncogenic RAS proteins have long been considered undruggable, due to an apparent lack of binding pockets suitable for small molecule inhibitors and the minute differences of RAS mutants from the wild-type proteins. 171580388v1

[0007] However, the discovery of covalent inhibitors targeting KRAS(G12C) in the GDP-bound state (Ostrem et al., “K-Ras(G12C) Inhibitors Allosterically Control GTP Affinity and Effector Interactions,” Nature 503:548-551 (2013); Janes et al., “Targeting KRAS Mutant Cancers with a Covalent G12C-Specific Inhibitor,” Cell 172:578-589 e517 (2018)) has revolutionized the field on two fronts: the revelation of a dynamic and druggable pocket under the switch II region, termed S-II pocket, and the effectiveness of mutant-selective targeting with G12C inhibitors (Canon et al., “The Clinical KRAS(G12C) Inhibitor AMG 510 Drives Anti- tumour Immunity,” Nature 575:217-223 (2019); Hallin et al., “The KRAS(G12C) Inhibitor MRTX849 Provides Insight toward Therapeutic Susceptibility of KRAS-Mutant Cancers in Mouse Models and Patients,” Cancer Discov 10:54-71 (2020)), followed by recent FDA approval of sotorasib and adagrasib. These discoveries have revised the notion that RAS is undruggable and sparked renewed enthusiasm for drug discovery against other oncogenic RAS mutant proteins.

[0008] RAS mutants other than G12C, for which the covalent inhibitor approach cannot be readily applied, still present formidable challenges for inhibitor discovery. Among these mutants, KRAS(G12D) is prevalent in particularly challenging cancers, including pancreatic and colorectal cancer (Prior et al., “The Frequency of Ras Mutations in Cancer,” Cancer Res 80:2969-2974 (2020)). Until recently, there were no reports of small molecule inhibitors selective to KRAS(G12D). Consequently, there have been intense efforts to develop KRAS(G12D)-selective inhibitors using polypeptides. There are two families of cyclic peptides that target KRAS(G12D) (Sakamoto et al. “Generation of KS-58 as the First K-Ras(G12D)- inhibitory Peptide Presenting Anti-cancer Activity in vivo,” Sci Rep 10:21671 (2020); Zhang et al., “GTP-State-Selective Cyclic Peptide Ligands of K-Ras(G12D) Block Its Interaction with Raf,” ACS Cent Sci 6:1753-1761 (2020)). Both classes of compounds bind in the S-II pocket. Sakamoto et al. have developed cyclic peptides that have ~4-fold selectivity for KRAS(G12D) over the wild-type (Sakamoto et al. “Generation of KS-58 as the First K-Ras(G12D)-inhibitory Peptide Presenting Anti-cancer Activity in vivo,” Sci Rep 10:21671 (2020)). These peptides can be derivatized into a cell-permeable form, which was shown to inhibit RAS-mediated signaling in cells and tumor growth in a mouse xenograft model. However, the low selectivity of these peptides and the presence of wild-type RAS proteins in cells (KRAS4A, KRAS4B, HRAS and NRAS) make it difficult to attribute the observed effects of these peptides solely to selective inhibition of the KRAS(G12D) allele in these cancer cells. Zhang et al. have developed cyclic peptides that bind dominantly to the GTP-bound state of KRAS(G12D) as well as those bind to both GTP- and GDP-bound states (Zhang et al., “GTP-State-Selective Cyclic Peptide Ligands of 171580388v1K-Ras(G12D) Block Its Interaction with Raf,” ACS Cent Sci 6:1753-1761 (2020)). However, these peptides have low affinity and minimal cell-permeability, and their efficacy in a cellular context has not been reported.

[0009] Recently, a small-molecule inhibitor that selectively targets KRAS(G12D), MRTX1133, has been developed (Wang et al., “Identification of MRTX1133, a Noncovalent, Potent, and Selective KRAS(G12D) Inhibitor,” J Med Chem 65:3123-3133 (2022); Hallin et al., “Anti-tumor Efficacy of a Potent and Selective Non-covalent KRAS(G12D) Inhibitor,” Nat Med 28:2171-2182 (2022)). The molecule appears effective at inhibiting the proliferation of cells harboring G12D mutation but not cells harboring wild-type KRAS. Unlike the G12C-selective drugs, the small molecule targeting G12D is noncovalent and its efficacy in the clinic will depend on several factors such as its pharmacokinetic profile in humans and therapeutic window for safe dosage. This impressive success has established the feasibility of selectively targeting KRAS(G12D) in a noncovalent manner. To comprehensively define how one can selectively target this prevalent KRAS mutation, it is still important to examine a diverse array of drug modalities against KRAS(G12D) and define the extent of plasticity of the S-II pocket.

[0010] The monobody system, which involves synthetic binding polypeptides derived from the fibronectin type III (FN3) domain, has been used to generate intracellular biologics targeting RAS and other proteins (Akkapeddi et al., “Monobodies as Tool Biologics for Accelerating Target Validation and Druggable Site Discovery,” RSC Med Chem 12:1839-1853 (2021)). Monobody inhibitors that are highly selective to KRAS(G12V) and KRAS(G12C) were recently reported, demonstrating the feasibility of achieving selective and noncovalent inhibition and degradation of these KRAS mutants (Teng et al., “Selective and Noncovalent Targeting of RAS Mutants for Inhibition and Degradation,” Nat Commun 12:2656 (2021)). It would be desirable, however, to develop monobody inhibitors that are highly selective toward KRAS(G12D) and potently inhibit KRAS(G12D)-mediated signaling, which can be used as intracellular biologics.

[0011] The present invention is directed to overcoming these and other deficiencies in the art. SUMMARY OF THE INVENTION

[0012] A first aspect relates to mutant KRAS binding polypeptide that includes a fibronectin type III (FN3) domain having one or more modified loop sequences comprising a modified FG loop amino acid sequence, a modified BC loop amino acid sequence, a modified DE loop amino acid sequence, or a combination thereof, and optionally one or more modified 171580388v1beta strand sequences, wherein said one or more modified loop sequences and said one or more optional modified beta strand sequences enable selective binding to a mutant KRAS comprising a G12D substitution at residue 12 of KRAS (KRASG12D) over wildtype KRASG12.

[0013] In certain embodiments, the binding polypeptide selectively binds to mutant KRASG12Dover a mutant KRAS comprising a G12C substitution at residue 12 of KRAS (KRASG12C) or a mutant KRAS comprising a G12V substitution at residue 12 of KRAS (KRASG12V).

[0014] A second aspect relates to a mutant KRAS binding polypeptide conjugate that includes a first portion that includes the mutant KRAS binding polypeptide according to the first aspect, and a second portion that is coupled to the first portion and is selected from the group of an E3 ubiquitin ligase ligand, an E3 ligase subunit, an E2 ubiquitin-conjugating enzyme, or an E2 recruiter.

[0015] In certain embodiments, the mutant KRAS binding polypeptide conjugate may also include a delivery vehicle.

[0016] A third aspect relates to a polynucleotide encoding the mutant KRAS binding polypeptide according to the first aspect or the mutant KRAS binding polypeptide conjugate according to the second aspect. Also encompassed by this aspect are vectors that include the polynucleotide as well as host cells that contain the polynucleotide or the vector.

[0017] A fourth aspect relates to a delivery vehicle that include the polynucleotide or the vector according to the third aspect.

[0018] A fifth aspect relates to a fusion construct that includes a first portion including a polynucleotide encoding the mutant KRAS binding polypeptide according to the first aspect (i.e., a polynucleotide according to the third aspect) and a second portion that is operatively coupled to the first portion and includes a polynucleotide encoding a E3 ligase subunit or an E2 ubiquitin- conjugating enzyme, or an imaging protein or peptide thereof.

[0019] A sixth aspect relates to a pharmaceutical composition that includes a pharmaceutically acceptable carrier and the mutant KRAS binding polypeptide according to the first aspect, the mutant KRAS binding polypeptide conjugate according to the second aspect, the polynucleotide and / or the vector according to the third aspect, the delivery vehicle according to the fourth aspect, or the fusion construct according to the fifth aspect.

[0020] A seventh aspect relates to a method for treating cancer in a subject. This method includes the step of administering to the subject having cancer a pharmaceutical composition according to the sixth aspect in an amount effective to treat the cancer. 171580388v1

[0021] An eighth aspect relates to a method for detecting the presence of KRAS(G12D) in a sample. This method includes the steps of contacting a sample with the mutant KRAS binding polypeptide according to the first aspect, and determining whether the mutant KRAS binding polypeptide specifically binds to KRAS(G12D).

[0022] A ninth aspect relates to a method for in vivo diagnostic imaging. This method includes the steps of administering a composition comprising the mutant KRAS binding polypeptide according to the first aspect under conditions effective to allow the mutant KRAS binding polypeptide to bind specifically to KRAS(G12D); and obtaining an image of a site in a patient where the mutant KRAS binding polypeptide has bound to KRAS(G12D).

[0023] A tenth aspect relates to a method for screening compounds for binding to KRAS(G12D). This method includes the steps of exposing KRAS(G12D) to the mutant KRAS binding polypeptide according to the first aspect and a compound being screened for binding activity; and determining whether the mutant KRAS binding polypeptide binds to KRAS(G12D) or the compound being screened binds to KRAS(G12D).

[0024] The accompanying Examples demonstrate the development and generation of multiple monobodies, synthetic binding proteins based on a fibronectin type III (FN3) scaffold, which are highly selective to KRAS(G12D). Their exceptional selectivity is based on three principles: first, they are selective toward KRAS over the other RAS isotypes (HRAS and NRAS); second, they are selected toward KRAS(G12D) over WT KRAS; and, third, in some clones, they are more selective toward the GTP-bound state over the GDP-bound state. These monobodies are selectively engaged with KRAS(G12D) when they are expressed in cultured cells as genetically encoded reagents. These monobodies in the purified protein format also selectively capture KRAS(G12D) from cell lysates.

[0025] Although intracellular biologics in the protein form are difficult to deliver into cells, similar to peptides, they can be coupled to delivery vehicles that promote cell uptake or readily delivered into cells as genetically encoded reagents using viruses, expression vectors and mRNA constructs. Furthermore, the KRASG12D-binding polypeptides disclosed herein can be used as the RAS-targeting moiety as a degrader fusion protein with a subunit of E3 ubiquitin ligases, such as VHL and SPOP (Teng et al., “Selective and Noncovalent Targeting of RAS Mutants for Inhibition and Degradation,” Nat Commun 12:2656 (2021); Simpson et al., “Inducible Degradation of Target Proteins through a Tractable Affinity-Directed Protein Missile System,” Cell Chem Biol 27:1164-1180 e1165 (2020); Bery et al., “A Potent KRAS Macromolecule Degrader Specifically Targeting Tumours with Mutant KRAS,” Nat Commun 11:3233 (2020); Lim et al., “Exquisitely Specific anti-KRAS Biodegraders Inform on the 171580388v1Cellular Prevalence of Nucleotide-Loaded States,” ACS Cent Sci 7:274-291 (2021), each of which is hereby incorporated by reference in its entirety). Thus, intracellular biologics such as monobodies are powerful tools for establishing the feasibility of selective targeting, elucidating the structural basis of specificity and characterizing effects of selective inhibition (and degradation) in cellular and organismal contexts. They will certainly prove useful for selective targeting of KRAS(G12D) in the treatment of KRAS(G12D)-mediated cancers. In addition, the KRASG12D-binding polypeptides disclosed herein can immediately be used for diagnostic purposes (i.e., screening for the presence of the KRAS(G12D) in patient samples or for in vivo diagnostics of KRAS(G12D)-mediated cancers) and in small molecule screening assays to detect small molecules the exhibit high affinity binding to KRAS(G12D). BRIEF DESCRIPTION OF THE DRAWINGS

[0026] FIGS.1A-1F illustrate activity of the KRAS(G12D)-selective monobody, 12D1. FIG.1A shows the binding titration of 12D1 displayed on the yeast cell surface to the indicated KRAS proteins. The mean (n = 3; technical replicates) of the median fluorescence intensity is shown. FIG.1B shows the specificity of 12D1 to different KRAS mutants bound to GTPγS (left) and GDP (right) at 100 nM. The bars indicate the means (n = 3, technical replicates). FIG.1C shows the BLI sensorgrams of the interaction between 12D1 and KRAS. The indicated KRAS proteins were immobilized and binding of a soluble 12D1 sample was measured. The KDvalues are from the global fit of a 1:1 binding model to the data. FIG.1D shows the inhibition by 12D1 of the binding of KRAS(G12D)•GTPγS to the Ras binding domain of c-Raf kinase protein (“RAF RBD”). Binding of 150 nM biotinylated KRAS(G12D)•GTPγS in complex with streptavidin-Dylight650 to RAF RBD immobilized on M270 Dynabeads in the presence of 0 nM–1000 nM 12D1. Complete inhibition (100% inhibition) is defined as the signal intensity in the absence of KRAS(G12D). FIG.1E shows that 12D1 does not bind to HRAS(G12D) in the absence of the KRAS-mimicking mutation, Q95H. FIG.1F shows that 12D1 does not bind to NRAS (G12D). Binding titration of the indicated HRAS and NRAS mutant samples to 12D1 displayed on the yeast cell surface is shown.

[0027] FIGS.2A-2E illustrate the crystal structure of 12D1(K63S) in complex with KRAS(G12D)•GDP, and comparisons with the structures of other KRAS(G12D) inhibitors. FIG.2A shows a comparison of 12D1(K63S)-KRAS(G12D)•GDP with MRTX1133- KRAS(G12D)•GDP (PBD: 7RPZ), KD2-KRAS(G12D)•GppNHp (PDB: 6WGN) and KRpep- 2d-KRAS(G12D)•GDP (PDB: 5XCO). In a color version of this figure, 12D1(K63S) and other inhibitors are depicted in blue, whereas the switch I and switch II regions and the G12D residue 171580388v1are shown in magenta, green and yellow, respectively. MRTX1133 is only marginally visible in this mode of presentation. FIG.2B shows close-up views of the G12D and Q61 residues (yellow in color version) and surrounding residues of inhibitors (cyan in color version) in the four structures. The hydrogen bonds are indicated as dashed lines.12D1(K63S) directly interacts with the side chain of the G12D mutation, which resembles interaction between MRTX1133 and the G12D mutation. FIG.2C shows the superpositions of other inhibitors (magenta in color version) on 12D1 residues (cyan in color version) in the S-II pocket. The structures were superimposed using residues 76-162 of KRAS as the reference. The b4 and a3 regions of KRAS are shown for orientation. FIG.2D shows a comparison of the backbone conformations of the switch I (magenta in color version) and switch II (green in color version) regions of KRAS in the four structures. The inhibitors are not shown for clarity. FIG.2E shows a comparison of the S-II pocket in the four structures. E62RAS, Y64RAS, and Q99RASresides are shown in light blue in color version, the nucleotides are shown in orange in color version, and Asp12 in yellow in color version. The distance between E62RASand Q99RAS(dotted line, yellow in color version) is indicated under each structure. For MRTX1133-KRAS(G12D)•GDP, the side chains of E62RAS, Y64RASand Q99RASare removed in order to expose the pocket.

[0028] FIGS.3A-3E illustrate the affinity maturation of KRAS(G12D)-selective monobodies. FIG.3A shows the amino acid sequences of monobody clones 12D1 (SEQ ID NO: 18), 12D2 (SEQ ID NO: 19), 12D3 (SEQ ID NO: 20), 12D4 (SEQ ID NO: 21), and 12D5 (SEQ ID NO: 23). In a color version of this figure, residues in red indicate mutations with respect to the parental scaffold. FIGS.3B-3D are BLI sensorgrams of 12D2, 12D3, and 12D4, respectively, interacting with immobilized KRAS. The KDvalue was determined from the global fit of the data with a 1:1 binding model. FIG.3E shows the capture of endogenous RAS proteins with 12D2. The “pull down” reaction was done with the indicated monobodies (12D2 and 12VC1) and RAF RBD, followed by immunoblotting using an anti-pan RAS antibody.12VC1 is selective to KRAS(G12C) and KRAS(G12V), and was used as a control.

[0029] FIGS.4A-4D illustrate the crystal structures of 12D5(K63S) in complex with KRAS(G12D)•GTPγS and with KRAS(G12D)•GDP. FIG.4A shows a comparison of 12D5(K63S)-KRAS(G12D)•GTPγS (left panel) and 12D5(K63S)-KRAS(G12D)•GDP (center panel) with 12D1(K63S)-KRAS(G12D)•GDP (right panel). In the color version of this figure, the color scheme is the same as in FIG.2. FIG.4B shows superpositions of 12D5(K63S)- KRAS(G12D)•GTPγS (left panel) and 12D1(K63S)-KRAS(G12D)•GDP (right panel) on 12D5(K63S)-KRAS(G12D)•GDP. FIG.4C shows close-up views of the G12DRASand Q61RASresidues (yellow in color version) and surrounding residues of 12D1 or 12D5 (cyan in color 171580388v1version). The hydrogen bonds are indicated as dashed lines. FIG.4D shows close-up views of the S-II pocket and surrounding residues of 12D1 or 12D5 (cyan in color version).

[0030] FIGS.5A-5F illustrate the deep mutational scanning analysis of 12D4 interface residues. FIG.5A shows flow cytometry profiles monitoring the binding of KRAS(G12D)•GTPγS (100 nM) to yeast cells displaying 12D4 (left), the deep mutational scanning library (middle) and sorted pools (right). The mutated positions in the 12D4 sequence are highlighted with the cyan boxes in color version. FIG.5B shows heat map representation of the numbers of reads for substitutions recovered in the three pools from library sorting with KRAS(G12D)•GTPγS. The number of reads for each substitution was normalized to the total number of reads for each entire pool and multiplied by 1000. The white squares indicate the wild-type residues. The locations of the residues in the monobody secondary structure elements are indicated on the bottom of the left panel. FIG.5C shows heat map representation of the numbers of reads for substitutions recovered in the three pools from library sorting with KRAS(G12D)•GDP. The data were analyzed in the same manner as in FIG.5B. The residue numbers in red (in color version) indicate positions whose amino acid profile differs between the pools sorted with KRAS(G12D)•GTPγS and KRAS(G12D)•GDP. FIGS.5D-5E show close-up views of K75Mband adjacent residues (5D), and of V29Mb,V30Mb, F31Mband adjacent residues (5E) in the 12D5-KRAS(G12D)•GTPγS complex. Ne of K75Mbinteracts with three backbone carbonyl groups of residues in the FG loop of 12D5, depicted with the dashed lines. In FIG.5F, the region corresponding to that depicted in FIG.5E of Darin K13 in complex with KRAS(WT) (PDB:6H46). The π-π stacking interaction between W35 of DARPin K13 and H95 of KRAS(WT) resembles the interaction between F31Mbof monobody 12D5 and H95 of KRAS(G12D) depicted in FIG.5E.

[0031] FIG.6 shows the selective engagement of KRAS(G12D) by monobodies. Colocalization of mCherry fused 12D2, 12D3, and 12D4 (pseudo-color magenta in color version) with overexpressed EGFP-fused KRAS(G12D) and KRAS(WT) (pseudo-color green in color version) in HEK293T cells. The scale bar denotes 10 mm. The graphs on the right show the fluorescence intensity profiles under the lines (yellow in color version) in the microscopy images.

[0032] FIGS.7A-7E show the crystal structure of 12D1(K63S) in complex with KRAS(G12D)•GDP (related to FIGS.2A-2D). FIG.7A shows the results of interpreting the electron density map of the region surrounding the bound nucleotide as GDP (left) and GTPgS (right). The 2mFo-DFc map contoured at 1 σ (light cyan mesh in color version) and mFo-DFc map with negative (red mesh in color version) and positive (green mesh in color version) density 171580388v1contoured at 3 σ are shown. The conformation of GDP and the Mg2+coordination are consistent with other KRAS(G12D)•GDP structures, and no negative density was found in this region (left). Negative density around β- and γ-phosphate of GTPγS was observed for GTPγS (right), and the conformation of GTPγS differs from that of GTP analogs in other KRAS(G12D) structures (see also FIG.10B). These observations justify interpretation of the electron density as GDP instead of GTPγS. FIG.7B shows the 2mFo-DFc composite omit map (contoured at 1 σ) of the binding interface around G12D (yellow in color version) and Q61 (yellow in color version) of KRAS(G12D) (gray in color version). The residues of monobody 12D1(K63S) are shown in cyan in color version. The water molecules (except for those coordinating with Mg2+) are omitted for clarity. FIG.7C shows the details of the interaction interface between 12D1(K63S) and KRAS(G12D). In a color version of this figure, the color scheme is the same as in FIG.2B. The C, D and F strands of 12D1 are labeled. The dashed lines indicate hydrogen bonds. FIG.7D shows a structural comparison of the 12D1(K63S)-KRAS(G12D)•GDP complex (left panel) with the 12VC1-HRAS(G12C)•GTPγS complex (PDB: 7L0G, right panel). Monobodies are shown in blue in color version and the RAS proteins are shown in gray in color version. G12D and G12C are shown in the stick model in yellow in color version. The beta-strands and the DE and FG loops of 12D1 are labeled. FIG.7E shows a close-up view of the interaction between H95RASand F31Mband T49Mb. The dashed line indicates a hydrogen bond.

[0033] FIGS.8A-8C show comparisons of the conformations of the switch I and switch II regions of KRAS. FIG.8A shows a comparison of 12D1(K63S)-bound KRAS(G12D)•GDP (left panel) with KRAS(G12D) •GppNHp (PDB: 5USJ, center panel) and KRAS(G12D)•GDP (PDB: 4EPR, right panel). The switch I and switch II regions are shown in magenta and green, respectively, in color version. Monobody 12D1(K63S) in the left panel is not shown for clarity. FIG.8B shows an overlay of 12D1(K63S)-bound KRAS(G12D)•GDP (top left) with other KRAS(G12D) structures. The switch I, switch II regions and the loop region between the b3 strand and the a2 helix in the 12D1(K63S)-bound KRAS(G12D)•GDP complex are shown in dark purple, dark green and orange, respectively, in color version. The color scheme for other KRAS(G12D) structures is the same as in FIG.8A. The loop region followed by the switch II region is shifted toward the switch I region upon binding of monobody 12D1(K63S), which likely causes the conformational shift of the switch I region. Indeed, the switch I region in other KRAS(G12D) structures would cause steric hindrance with the switch II region observed in the 12D1(K63S)-bound KRAS(G12D)•GDP complex, as shown in the overlaid structures. FIG.8C shows crystal packing near the switch I and switch II regions of the KRAS(G12D)•GDP in complex with 12D1(K63S). The proteins in the asymmetric unit (ASU) and the symmetric unit 171580388v1are shown in gray and cyan, respectively, in color version. The switch I and switch II regions in the complex in the ASU are shown in magenta and green, respectively, in color version. The dashed lines indicate polar interactions.

[0034] FIGS.9A-9I show yeast display characterization of KRAS(G12D)-selective monobodies, 12D2, 12D3 and 12D4. FIG.9A shows binding titrations of KRAS(G12D) and KRAS(WT) to yeast cells displaying the 12D2 monobody. FIG.9B shows specificity of 12D2 to different KRAS mutants in the GTPγS- and GDP-bound states. FIG.9C shows binding titrations of HRAS(G12D) and HRAS(G12D:Q95H) to yeast cells displaying 12D2. FIG.9D shows binding titrations of KRAS(G12D) and KRAS(WT) to yeast cells displaying the 12D3 monobody. FIG.9E shows specificity of 12D3 to different KRAS mutants in the GTPγS- and GDP-bound states. FIG.9F shows binding titrations of HRAS(G12D) and HRAS(G12D:Q95H) to yeast cells displaying 12D3. FIG.9G shows binding titrations of KRAS(G12D) and KRAS(WT) to yeast cells displaying the 12D4 monobody. FIG.9H shows specificity of 12D4 to different KRAS mutants in the GTPγS- and GDP-bound states. FIG.9I shows binding titrations of HRAS(G12D) and HRAS(G12D:Q95H) to yeast cells displaying 12D4. Data shown are the mean and s.d. (n = 3).

[0035] FIGS.10A-10C show binding characterization of the 12D5 monobody and crystal structures of 12D5 in complex with KRAS(G12D)•GTPγS and with KRAS(G12D)•GDP. FIG. 10A shows BLI sensorgrams of the interaction between 12D5 and KRAS. The indicated KRAS proteins were immobilized and the binding of the soluble 12D5 sample was measured. The KDvalues are from the global fit of a 1:1 binding model to the data. FIG.10B shows the 2mFo-DFc and mFo-DFc difference maps of the region surrounding the bound nucleotide of two KRAS molecules in the asymmetric unit. The maps are shown in the same manner as FIG.7A. The conformation of nucleotides and Mg2+coordination are consistent with other KRAS(G12D) structures. No negative density was observed in these regions. FIG.10C shows the conformations of two KRAS molecules found in the asymmetric unit of 12D5-bound KRAS(G12D). The color scheme is the same as in FIGS.2A-2D. KRAS is shown as cartoon with the D12RASside chains and bound nucleotides in stick models. Only the residues of 12D5 monobody surrounding D12RASare shown in cyan (in color version) for clarity.

[0036] FIGS.11A-11C show incompatibility of the 12D5-KRAS(G12D)•GTPγS complex with binding to natural RAS-binding partners. FIG.11A shows an overlay of the 12D5- RAS(G12D) complex and a RAF RBD-KRAS(WT) complex (PBD: 6XHB) using the RAS molecules as the references. RAF RBD-bound KRAS(WT) is not shown for clarity. The box shows the steric hindrance between the switch regions of KRAS(G12D) and RAF RBD (green in 171580388v1color version). FIG.11B shows an overlay of the 12D5-RAS complex and an RAS-PIK3γ RBD complex (PBD: 1HE8) in the same manner as in FIG.11A. FIG.11C shows an overlay of the 12D5-RAS complex and a SOS-HRAS•GppNHp complex (PDB: 1NVV) in the same manner as in FIG.11A. HRAS• GppNHp is not shown for clarity. DETAILED DESCRIPTION OF THE INVENTION

[0037] The present invention relates generally to KRAS(G12D)-binding polypeptides, polynucleotides encoding the KRAS(G12D)-binding polypeptides, conjugates that include the KRAS(G12D)-binding polypeptide, and polynucleotides encoding the KRAS(G12D)-binding polypeptides and conjugates. The disclosure also relates to methods of using these KRAS(G12D)-binding polypeptides, conjugates, and polynucleotides encoding the same for the treatment of oncogenic KRAS(G12D)-mediated cancers and related conditions, for diagnostic purposes in identifying the presence of KRAS(G12D), and for competitive binding assays to identify small molecules that also demonstrate improved properties in binding specifically to KRAS(G12D).

[0038] Accordingly, a first aspect of the disclosure relates to a KRAS(G12D)-binding polypeptide (also referred to herein as a “mutant KRAS binding polypeptide”). This mutant KRAS binding polypeptide includes an FN3 domain having one or more of a modified FG loop amino acid sequence, a modified BC loop amino acid sequence, a modified DE loop amino acid sequence, or any combination of the aforementioned modified loop sequences, and optionally one or more modified beta strand amino acid sequences. The one or more modified loop sequences and the one or more optional modified beta strand sequences enable selective binding to a mutant KRAS comprising a G12D substitution at residue 12 of KRAS (KRASG12Dor KRAS(G12D)) over wildtype KRAS.

[0039] As demonstrated herein the mutant KRAS binding polypeptides bind specifically to KRASG12Dmutant protein. The selective binding of the mutant KRAS binding polypeptides to KRASG12Dover wildtype KRAS is such that the affinity for KRASG12Dis at least 10-fold greater than the affinity for wildtype KRAS, preferably at least 20-fold greater, at least 30-fold greater, or at least 40-fold greater than the affinity for wildtype KRAS. In certain embodiments, the he affinity for KRASG12Dis at least 50-fold greater, at least 75-fold greater, or at least 100- fold greater than the affinity for wildtype KRAS.

[0040] The FN3 domain is an evolutionary conserved protein domain that is about 100 amino acids in length and possesses a beta sandwich structure. The beta sandwich structure of human FN3 comprises seven beta-strands, referred to as strands A, B, C, D, E, F, G, with six 171580388v1connecting loops, referred to as loops AB, BC, CD, DE, EF, and FG that exhibit structural homology to immunoglobulin binding domains. Three of the six loops, i.e., loops DE, BC, and FG, correspond topologically to the complementarity determining regions of an antibody, i.e., CDR1, CDR2, and CDR3. The remaining three loops are surface exposed in a manner similar to antibody CDR3. In accordance with the present disclosure, one or more of the loop regions and, optionally, one or more beta strands of each FN3 domain of the binding molecule are modified to enable specific binding to mutant KRASG12Dprotein.

[0041] As used herein “specifically binds” or “specific binding” refers to the ability of the FN3 containing binding molecule of the disclosure to bind to a predetermined antigen, i.e., mutant KRASG12Dwith a dissociation constant (KD) of about 1×10-6M or less, for example about 1×10-7M or less, about 1×10-8M or less, about 1×10-9M or less, about 1×10-10M or less, about 1×10-11M or less, about 1×10-12M or less, or about 1×10-13M or less. Typically, the FN3 domain binds to mutant KRASG12Dwith a KD that is at least ten-fold less than its KD for a nonspecific antigen (for example BSA or casein), and in particular wildtype KRAS, as measured by surface plasmon resonance using for example a Proteon Instrument (BioRad) or by biolayer interferometry using for example an Octet instrument (Sartorius). Thus, the KRAS(G12D)- binding polypeptides have greater affinity for mutant KRASG12Dthan wildtype KRAS and KRAS having other mutations at position 12 (i.e., KRASG12Cor KRASG12V).

[0042] The modified FN3 domain of the binding molecule of the present disclosure can be a FN3 domain derived from any of the wide variety of animal, yeast, plant, and bacterial extracellular proteins containing these domains. In one embodiment, the FN3 domain is derived from a mammalian FN3 domain. Exemplary FN3 domains include, for example and without limitation, any one of the 15 different FN3 domains present in human tenascin C, or the 15 different FN3 domains present in human fibronectin (FN), for example, the 10th fibronectin type III domain. Exemplary FN3 domains also include non-natural synthetic FN3 domains, such as those described in U.S. Pat. Publ. No.2010 / 0216708 to Jacobs et al., which is hereby incorporated by reference in its entirety. Individual FN3 domains are referred to by domain number and protein name, e.g., the 10th FN3 domain of fibronectin (10FN3).

[0043] In some embodiments, the FN3 domain of the binding molecule is derived from the 10th FN domain of fibronectin (10FN3). In some embodiments, the FN3 domain of the binding molecule is derived from the human 10FN3 domain. The human 10FN3 domain has the amino acid sequence of SEQ ID NO:1 as shown below. The locations of the BC (residues 24- 30), CD (residues 40-45), DE (residues 51-55), and FG (residues 75-86) loops are underlined 171580388v1within the wild-type sequence of SEQ ID NO: 1. Locations of other amino acid residues referenced in this disclosure are also identified within SEQ ID NO: 1 by their position. VSDVPRDLEVVAATPTSLLISWDA24PAVTVR30Y31YR33ITYGETG40GNSPV45QE47FT49V P51GSKS55TATISGLKPGVDYTITVYAV75TGRGDSPASSK86PISINYRT (SEQ ID NO: 1)

[0044] In accordance with the present disclosure, one or more of the loop regions or selected residues within one or more of these loop regions are modified to enable mutant KRASG12Dbinding specificity and affinity. Suitable modifications include amino acid residue substitutions, insertions, and / or deletions. In one aspect, amino acid residues in at least one, at least two, at least three, at least four, at least five, or all six of the loop regions are altered for mutant KRASG12Dbinding specificity and affinity. In one embodiment, one or more amino acid modifications within the loop regions at or about residues 24-30 (BC loop), 40-45 (CD loop), 51- 55 (DE loop), and / or 75-86 (FG loop) of SEQ ID NO:1 form the mutant KRASG12Dbinding region. In another embodiment, one or more amino acid modification within any one of these loop regions enable mutant KRASG12Dbinding.

[0045] In some embodiments, the mutant KRASG12Dbinding polypeptide of the present disclosure comprises a modified BC loop. In some embodiments, the modified BC loop includes the amino acid sequence of XXXVTXV (SEQ ID NO: 17), where X is any amino acid residue. In some embodiments, the modified BC loop is APAVTVV (SEQ ID NO: 13) (see Table 1), or a BC loop having one, two, or three substitutions or an insertion to SEQ ID NO: 13. In certain embodiments, the substitution can be a conserved substitution. In other embodiments, the substitution can be a non-conserved substitution.

[0046] In some embodiments, the mutant KRASG12Dbinding polypeptide of the present disclosure includes a modified DE loop. In some embodiments, the modified DE loop includes the amino acid sequence of X1GSX4X5, (SEQ ID NO: 11), where each of X4and X5is any amino acid residue, and X1is any amino acid residue, but preferably Pro or Ser. In some embodiments, the modified DE loop includes the amino acid sequence of PGSKS (SEQ ID NO: 7) or PGSKS (SEQ ID NO: 8) (see Table 1), or a DE loop having one, two, or three substitutions or an insertion to SEQ ID NO: 7 or 8. In certain embodiments, the substitution can be a conserved substitution. In other embodiments, the substitution can be a non-conserved substitution.

[0047] In some embodiments, the mutant KRASG12Dbinding polypeptide of the present disclosure includes a modified FG loop. In some embodiments, the modified FG loop includes the amino acid sequence of KYLX4X5X6GYX8X9, (SEQ ID NO: 6), where X4is F or W, X5is W 171580388v1or Y, X6is S or K, X8is any amino acid residue but preferably S, and X9is any amino acid residue but preferably S. In some embodiments, the modified FG loop includes the amino acid sequence of KYLWYSGYSS (SEQ ID NO: 2), KYLWYSGYSS (SEQ ID NO: 3), KYLFWKGYSS (SEQ ID NO: 4), or KYLFWSGYSS (SEQ ID NO: 5) (see Table 1), or an FG loop having one, two, or three substitutions or an insertion to any one of SEQ ID NOS: 2 to 5. In certain embodiments, the substitution can be a conserved substitution. In other embodiments, the substitution can be a non-conserved substitution. Table 1 – KRASG12DMonobody Loop Sequences

[0048] In various embodiments described above, the amino acid substitution at the one or more identified positions of the mutant KRASG12Dbinding polypeptide is a conservative substitution. Such conservative substitutions involve substituting one amino acid residue for another that is a member of the same class, which acts as a functional equivalent, resulting in a silent (or substantially silent) alteration. That is to say, the change relative to the explicitly disclosed sequence would not appreciably diminish the basic properties of mutant KRASG12Dbinding polypeptide. These classes of amino acid residues include, nonpolar (hydrophobic) amino acids (e.g., alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine); polar neutral amino acids (e.g., glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine); positively charged (basic) amino acids (e.g., arginine, lysine and histidine; and negatively charged (acidic) amino acids (e.g., aspartic acid and glutamic acid).

[0049] In other embodiments, an amino acid substitution at the one or more identified positions of the mutant KRASG12Dbinding polypeptide as described herein is a non-conservative alteration (i.e., a substitution that disrupts the sequence, structure, function, or activity of the identified region). A non-conservative substitution involves the substitution of an amino acid residue of one particular class with an amino acid residue of a different class. For example, a substitution of a nonpolar (hydrophobic) amino acid residue with a polar neutral amino acid or vice versa. In another embodiment, the non-conservative substitution involves the substitution of a positively charged (basic) amino acid residue, with a negatively charged (acidic) amino acid 171580388v1residue, such as aspartic acid and glutamic acid or vice versa. Such molecular alterations can be accomplished by methods well known in the art, including primer extension on a plasmid template using single stranded templates (Kunkel et al., Proc. Acad. Sci., USA 82:488-492 (1985), which is hereby incorporated by reference in its entirety), double stranded DNA templates (Papworth et al., Strategies 9(3):3-4 (1996), which is hereby incorporated by reference in its entirety), and by PCR cloning (Braman, J., ed., In Vitro Mutagenesis Protocols, 2nd ed. Humana Press, Totowa, N.J. (2002), which is hereby incorporated by reference in its entirety).

[0050] Where mutational scanning indicates that diverse classes of amino acids are tolerated at a particular position, then both conserved and non-conserved substitutions are contemplated. Where mutation scanning indicates that only specific substitutions are available at a particular position, then only those tolerated substitutions are contemplated for those positions.

[0051] Compared to previously identified FN3 domains that bind specifically and selectively to a mutant KRAS comprising a G12C / V substitution at residue 12 of KRAS (KRASG12Cor KRASG12V), but not to KRASG12D, the FN3 domains disclosed herein are not only functionally distinct but also structurally distinct. In particular, the FN3 domains disclosed herein possess structurally distinct CD and / or FG loop amino acid sequences.

[0052] As discussed above, FN3 domains contain two sets of CDR-like loops on the opposite faces of the molecule. The two sets of loops are separated by beta-strands (regions of the domain that are between the loops) that form the center of the FN3 structure. Like the loops, these beta-strands can be altered to enhance target molecule binding specificity and affinity. Preferably, some or all of the surface exposed residues in the beta strands are randomized without affecting (or minimally affecting) the inherent stability of the FN3 domain. In some embodiments, one or more of residues in one or more beta-strands is modified to enable interaction with mutant KRASG12D. Suitable modifications include amino acid substitutions, insertions, and / or deletions. For example, one or more amino acid residues of the A beta strand, the B beta strand, the C beta strand, the D beta strand, the E beta strand, the F beta strand, or the G beta strand may be modified to enable mutant KRASG12Dbinding or to enhance the specificity or affinity of mutant KRASG12Dbinding. In certain embodiments, the beta strand substitutions are conserved substitutions as described above. In other embodiments, the beta strand substitutions are non-conserved substitutions as described above.

[0053] In certain embodiments, one or more amino acid residues of the C and / or D beta- strands are modified for binding to the mutant KRASG12D. 171580388v1

[0054] In some embodiments, the mutant KRASG12Dbinding polypeptide described herein comprises one or more amino acid residue substitutions, additions, or deletions in the A beta strand or region upstream thereof.

[0055] In some embodiments, the mutant KRASG12Dbinding polypeptide comprises an amino acid substitution at one or more residues corresponding to residues Y31 and R33 of SEQ ID NO: 1. In some embodiments, the Y31 and / or R33 substitution is one of the substitutions listed in Table A or B in the accompanying Examples. In some embodiments, the amino acid substitution is tyrosine to phenylalanine substitution at the amino acid residue corresponding to the tyrosine at position 31 (Y31F) of SEQ ID NO: 1. In some embodiments, the amino acid substitution is arginine to aspartic acid substitution at the amino acid residue corresponding to the arginine at position 33 (R33D) of SEQ ID NO: 1.

[0056] In some embodiments, the mutant KRASG12Dbinding polypeptide comprises an amino acid substitution at one or more residues corresponding to residues E47 and R49 of SEQ ID NO: 1. In some embodiments, the E47 and / or R49 substitution is one of the substitutions listed in Table A or B in the accompanying Examples.

[0057] In some embodiments, the mutant KRASG12Dbinding polypeptide comprises a C- terminal extension, with a sequence, for example, of EIDK (SEQ ID NO: 22) at the C-terminal end.

[0058] In some embodiments, the mutant KRASG12Dbinding polypeptide comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 18. In some embodiments, the mutant KRASG12Dbinding polypeptide comprises an amino acid sequence that is at least 90% identical (such as at least 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical) to the amino acid sequence of SEQ ID NO: 18. In some embodiments, the mutant KRASG12Dbinding polypeptide comprises the amino acid sequence of SEQ ID NO: 18. SEQ ID NO: 18 Mb(KRAS_12D1) VSSVPTKLEVVAATPTSLLISWDAPAVTVVFYDITYGETGGNSPVQEFTVPGSKSTATISGLKP GVDYTITVYAKYLWYSGYSSPISINYRT

[0059] In some embodiments, the mutant KRASG12Dbinding polypeptide comprises an amino acid sequence that is at least 80% identical to an amino acid sequence of SEQ ID NO: 19. In some embodiments, the mutant KRASG12Dbinding polypeptide comprises an amino acid sequence that is at least 90% identical (such as at least 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical) to the amino acid sequence of SEQ ID NO: 19. In some embodiments, the mutant KRASG12Dbinding polypeptide comprises the amino acid sequence of SEQ ID NO: 19 (with mutations that enhanced the affinity in italics). 171580388v1SEQ ID NO: 19 Mb

[0060] In some embodiments, the mutant KRASG12Dbinding polypeptide comprises an amino acid sequence that is at least 80% identical to an amino acid sequence of SEQ ID NO: 20. In some embodiments, the mutant KRASG12Dbinding polypeptide comprises an amino acid sequence that is at least 90% identical (such as at least 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical) to the amino acid sequence of SEQ ID NO: 20. In some embodiments, the mutant KRASG12Dbinding polypeptide comprises the amino acid sequence of SEQ ID NO: 20 (with mutations that enhanced the affinity in italics). SEQ ID NO: 20 Mb(KRAS_12D3) VSSVPTKLEVVAATPTSLLVSWDAPAVTVVFYDITYGETGGNSPVQEFTVSGSKSTATISGLKP GVDYTITVYAKYLFWKGYSSPISINYRT

[0061] In some embodiments, the mutant KRASG12Dbinding polypeptide comprises an amino acid sequence that is at least 80% identical to an amino acid sequence of SEQ ID NO: 21. In some embodiments, the mutant KRASG12Dbinding polypeptide comprises an amino acid sequence that is at least 90% identical (such as at least 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical) to the amino acid sequence of SEQ ID NO: 21. In some embodiments, the mutant KRASG12Dbinding polypeptide comprises the amino acid sequence of SEQ ID NO: 21 (with mutations that enhanced the affinity in italics). SEQ ID NO: 21 Mb(KRAS_12D4) VSSVPTKLEVVAATPTSLLVSWDAPAVTVVFYDITYGETGGNSPVQEFTVSGSKSTATISGLKP GVDYTITVYAKYLFWSGYSSPISINYRT

[0062] In some embodiments, the mutant KRASG12Dbinding polypeptide comprises an amino acid sequence that is at least 80% identical to an amino acid sequence of SEQ ID NO: 23. In some embodiments, the mutant KRASG12Dbinding polypeptide comprises an amino acid sequence that is at least 90% identical (such as at least 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical) the amino acid sequence of SEQ ID NO: 23. In some embodiments, the mutant KRASG12Dbinding polypeptide comprises an amino acid sequence of SEQ ID NO: 23 (with mutations that enhanced the affinity in italics). 171580388v1SEQ ID NO: 23 Mb(KRAS_12D5) VSSVPTKLEVVAATPTSLLVSWDAPAVTVVFYDITYGETGGNSPVQEFTVPGSKSTATISGLKP GVDYTITVYAKYLFWSGYSSPISINYRTEIDK SEQ ID NO: 23 is one example of a mutant KRASG12Dbinding polypeptide having the C-terminal extension of EIDK (SEQ ID NO: 22).

[0063] In each of the above-identified monobodies, the C beta strand comprises two point mutations relative to the wildtype 10FN3 of SEQ ID NO: 1. Specifically, the C beta strand includes a Y31F substitution and a R33D substitution. Further modifications of these amino acid sequences are also contemplated, including any one or more of the alternative Y31 substitutions listed in Tables A-B, any one or more of the alternative R33 substitutions listed in Tables A-B, any one or more of the alternative E47 substitutions listed in Tables A-B, and any one or more of the alternative T49 substitutions listed in Tables A-B. In addition, C-terminal extension with EIDK may be useful. For example, Mb(KRAS_12D5) may include S51P and a C-terminal extension having the amino acid sequence EIDK (SEQ ID NO: 22).

[0064] Another aspect of the present disclosure relates to a mutant KRASG12Dbinding peptide conjugate comprising a first portion and a second portion. The first portion of the conjugate comprises the mutant KRASG12Dbinding polypeptide as described supra. The second portion of the conjugate, which is coupled to the first portion of the conjugate, is selected from a diagnostic moiety, an E3 ligase subunit, an E3 ubiquitin ligase binding ligand, an E2 ubiquitin- conjugating enzyme, and E2 recruiters such as N-((3-(3-fluoro-4-methoxyphenyl)-4,5- dihydroisoxazol-5-yl)methyl)acrylamide (also known as EN67) (see Forte et al., ACS Chem. Biol.18(4): 897–904 (2023), which is hereby incorporated by reference in its entirety).

[0065] In accordance with this aspect of the present disclosure, the first and second portions of the conjugate are covalently coupled to each other directly or via a linker. The first and second portions may be directly fused and generated by standard cloning and expression techniques. Alternatively, well known chemical coupling methods may be used to attach the portions directly or via a peptide or other linker to produce mutant KRASG12Dbinding peptide conjugates as described herein. For example, covalent conjugation of the first and second portions can be accomplished via lysine side chains using an activated ester or isothiocyanate, or via cysteine side chains with a maleimide, haloacetyl derivative or activated disulfide. Site specific conjugation of the first and second portions can also be accomplished by incorporating unnatural amino acids, self-labeling tags (e.g., SNAP or DHFR), or a tag that is recognized and modified specifically by another enzyme such as sortase A, lipoic acid ligase, and formylglycine- 171580388v1generating enzyme. In some embodiments, site specific conjugation of the first and second portions is achieved by the introduction of cysteine residue either at the C-terminus of the mutant KRASG12Dbinding molecule or at a specific site as described by Goldberg et al., “Engineering a Targeted Delivery Platform Using Centyrins,” Protein Engineering, Design & Selection 29(12):563-572 (2016), which is hereby incorporated by reference in its entirety.

[0066] In some embodiments, the first and second portions of the mutant KRASG12Dbinding peptide conjugate are coupled together via a linker. In some embodiments, the linker is an amino acid linker. In some embodiments, the amino acid linker is a cleavable linker. In some embodiments, the amino acid linker is a non-cleavable linker. Suitable linkers include peptides composed of repetitive modules of one or more of the amino acids, such as glycine and serine or alanine and proline. Exemplary linker peptides include, e.g., (Gly-Gly)n, (Gly-Ser)n, (Gly3- Ser)n, (Ala-Pro)n wherein n is an integer from 1-25. The length of the linker may be appropriately adjusted as long as it does not affect the function of the non-binding protein-drug conjugate. The standard 15 amino acid (Gly4-Ser)3linker peptide has been well-characterized and has been shown to adopt an unstructured, flexible conformation. In addition, this linker peptide does not interfere with assembly and activity of the domains it connects (Freund et al., “Characterization of the Linker Peptide of the Single-Chain Fv Fragment of an Antibody by NMR Spectroscopy,” FEBS 320:97 (1993), the disclosure of which is hereby incorporated by reference in its entirety).

[0067] In some embodiments, the second portion of the mutant KRASG12Dbinding peptide conjugate is a E3 ubiquitin ligase subunit. Exemplary E3 ubiquitin ligase subunits include, without limitation, those selected from the group of von Hippel-Lindau (VHL); cereblon, XIAP, E3A; MDM2; Anaphase-promoting complex (APC); UBR5 (EDD1); SOCS / BC-box / eloBC / CUL5 / RING; LNXp80; CBX4; CBLL1; HACE1; HECTD1; HECTD2; HECTD3; HECW1; HECW2; HERC1; HERC2; HERC3; HERC4; HUWE1; ITCH; NEDD4; NEDD4L; PPIL2; PRPF19; PIAS1; PIAS2; PIAS3; PIAS4; RANBP2; RNF4; RBX1; SMURF1; SMURF2; STUB1; TOPOR5; TRIP12; UBE3A; UBE3B; UBE3C; UBE4A; UBE4B; UBOX5; UBR5; WWP1; WWP2; Parkin; A20 / TNFAIP3; AMFR / gp78; ARA54; beta-TrCP1 / BTRC; BRCA1; CBL; CHIP / STUB1; E6; E6AP / UBE3A; F-box protein 15 / FBXO15; FBXW7 / Cdc4; GRAII / RNF128; HOIP / RNF31; cIAP-1 / HIAP-2; cIAP-2 / HIAP-1; cIAP (pan); ITCH / AIP4; KAP1; MARCH8; Mind Bomb 1 / MIB1; Mind Bomb 2 / MIB2; MuRF1 / TRIM63; NDFIP1; NEDD4; NleL; Parkin; RNF2; RNF4; RNF8; RNF168; RNF43; SART1; Skp2; SMURF2; TRAF-1; TRAF-2; TRAF-3; TRAF-4; TRAF-5; TRAF-6; TRIM5; TRIM21; TRIM32; UBR5; 171580388v1and ZNRF3. This listing above is by no means exhaustive, and it is contemplated that other E3 ubiquitin ligase subunits can be used as the second portion of the conjugate.

[0068] In another embodiment, the second portion of the mutant KRASG12Dbinding peptide conjugate is a E3 ubiquitin ligase ligand, which can be a small molecule ligand or peptide ligand.

[0069] Exemplary E3 ubiquitin ligase ligands include, without limitation, small molecule ligands such as thalidomide, lenalidomide, pomalidomide and analogs thereof known to bind to cereblon; cereblon ligands disclosed in US2016 / 0058872 and US2015 / 0291562, which are hereby incorporated by reference in their entirety; E3 ligase ligands that bind MDM2 include nutlin compounds such as nutlin 3a and nutlin 3, as well as those disclosed in any one of WO2012 / 121361; WO2014 / 038606; WO2010 / 082612; WO2014 / 044401; WO2009 / 151069; WO2008 / 072655; WO2014 / 100065; WO2014 / 100071; WO2014 / 123882; WO2014 / 120748; WO2013 / 096150; WO2015 / 161032; WO2012 / 155066; WO2012 / 065022; WO2011 / 060049; WO2008 / 036168; WO2006 / 091646; WO2012 / 155066; WO2012 / 065022; WO2011 / 153509; WO2013 / 049250; WO2014 / 151863; WO2014 / 130470; WO2014 / 134207; WO2014 / 200937; WO2015 / 070224; WO2015 / 158648; WO2014 / 082889; WO2013 / 178570; WO2013 / 135648; WO2012 / 116989; WO2012 / 076513; WO2012 / 038307; WO2012 / 034954; WO2012 / 022707; WO2012 / 007409; WO2011 / 134925; WO2011 / 098398; WO2011 / 101297; WO2011 / 067185; WO2011 / 061139; WO2011 / 045257; WO2010 / 121995; WO2010 / 091979; WO2010 / 094622; WO2010 / 084097; WO2009 / 115425; WO2009 / 080488; WO2009 / 077357; WO2009 / 047161A1; WO2008 / 141975A1, all of which are hereby incorporated by reference in their entirety; E3 ligase ligands that bind IAP include compounds disclosed in U.S. Pat. No.9,096,544; WO 2015187998; WO 2015071393; U.S. Pat. Nos.9,278,978; 9,249,151; US 20160024055; US 20150307499; US 20140135270; US 20150284427; US 20150259359; US 20150266879; US 20150246882; US 20150252072; US 20150225449; U.S. Pat. No.8,883,771, which are hereby incorporated by reference in their entirety; and E3 ligase ligands that bind SPOP or VHL such as hydroxyproline compounds disclosed in WO2013 / 106643, and other compounds described in US2016 / 0045607; WO2014187777; US20140356322; U.S. Pat. No.9,249,153; Yang et al., “Discovery of Thalidomide-based PROTAC Small Molecules as the Highly Efficient SHP2 Degraders,” Eur J Med Chem.218:113341 (2021); Crew et al., “Identification and Characterization of Von Hippel-Lindau-recruiting Proteolysis Targeting Chimeras (PROTACs) of TANK-binding Kinase 1,” J Med Chem.61(2):583–98 (2018); Henning et al., “Deubiquitinase-Targeting Chimeras for Targeted Protein Stabilization,” Nature Chem Biol. 18:412–421 (2022); Roth et al., “Targeting Endogenous K-RAS for Degradation through the 171580388v1Affinity-Directed Protein Missile System,” Cell Chem Biol 27:1151-1163 e1156 (2020); Teng et al., “Selective and Noncovalent Targeting of RAS Mutants for Inhibition and Degradation,” Nat Commun 12:2656 (2021); Lim et al., “Exquisitely Specific anti-KRAS Biodegraders Inform on the Cellular Prevalence of Nucleotide-Loaded States,” ACS Cent Sci 7:274-291 (2021); Fulcher et al., “Targeting Endogenous Proteins for Degradation Through the Affinity-directed Protein Missile System,” Open Biol 7 (2017), each of which is hereby incorporated by reference in its entirety. This listing above is by no means exhaustive, and it is contemplated that other E3 ligase ligands can be used as the second portion of the conjugate.

[0070] In some embodiments, the second portion of the mutant KRASG12Dbinding peptide conjugate is an E2 ubiquitin-conjugating enzyme. Exemplary E2 ubiquitin-conjugating enzymes include, without limitation, UBE2A, UBE2B, UBE2C, UBE2D1, UBE2D2, UBE2D3, UBE2D4, UBE2E1, UBE2E2, UBE2E3, UBE2E4P, UBE2F, UBE2G1, UBE2G2, UBE2H, UBE2I, UBE2J1, UBE2J2, UBE2K, UBE2L1, UBE2L2, UBE2L3, UBE2L4, UBE2L5, UBE2L6, UBE2M, UBE2N, UBE2NL, UBE2Q1, UBE2Q2, CDC34, UBE2R2, UBE2S, UBE2T, UBE2U, UBE2V1, UBE2V2, UBE2W, and UBE2Z. This listing above is by no means exhaustive, and it is contemplated that other E2 ubiquitin-conjugating enzymes can be used as the second portion of the conjugate.

[0071] In another embodiment, the second portion of the mutant KRAS binding peptide conjugate comprises a second polypeptide. In some embodiments, the second polypeptide is a non-binding molecule. In some embodiments, the polypeptide is a second binding molecule. In some embodiments, the second binding molecule is an antibody or antibody binding domain thereof.

[0072] As used herein, an antibody includes any protein or peptide containing molecule that comprises at least a portion of an immunoglobulin molecule, such as but not limited to, at least one, at least two, or at least three complementarity determining region (CDR) of a heavy or light chain, a heavy chain or light chain variable region, a heavy chain or light chain constant region, a framework region, or any portion thereof. Antibodies encompass full antibodies, digestion fragments, specified portions and variants thereof, including, without limitation, portions of antibodies that mimic the structure and / or function of an antibody or specified fragment or portion thereof, including, without limitation, single chain antibodies, single domain antibodies (i.e., antibody fragments comprising merely one variable domain, which might be VHH, VH or VL, that specifically bind an antigen or epitope independently of other V regions or domains). Functional fragments include antigen-binding fragments that bind to a particular target. For example, antibody fragments capable of binding to a particular target or portions 171580388v1thereof, include, but are not limited to, Fab (e.g., by papain digestion), Fab′ (e.g., by pepsin digestion and partial reduction) and F(ab′)2 (e.g., by pepsin digestion), Fd (e.g., by pepsin digestion, partial reduction and reaggregation), Fv or scFv (e.g., by molecular biology techniques) fragments.

[0073] In any embodiment, the pharmaceutically active moiety of the mutant KRASG12Dbinding peptide conjugate is optionally coupled to or packaged within a delivery vehicle. In certain embodiments, the delivery vehicle is a third portion of the conjugate, which is linked in the manner described above to the first and / or second portions of the conjugate. Accordingly, in some embodiments, the mutant KRASG12Dbinding peptide conjugate comprises the mutant KRASG12Dbinding polypeptide coupled to a delivery vehicle. In any embodiment, the delivery vehicle contains a pharmaceutically active moiety.

[0074] In accordance with this aspect of the disclosure, any suitable drug delivery vehicle known in the art can be coupled to the mutant KRASG12Dbinding polypeptide to form the mutant KRASG12Dbinding peptide conjugate as described herein. By way of example, the drug delivery vehicle can be a peptide or polypeptide responsible for cell uptake, a nanoparticle delivery vehicle, a polymer-based particle, or a lipid-based particle delivery vehicle known in the art (see, e.g., Xiao et al., “Engineering Nanoparticles for Targeted Delivery of Nucleic Acid Therapeutics in Tumor,” Mol. Ther. Meth. Clin. Dev.12:1-18 (2019); Ni et al., “Synthetic Approaches for Nucleic Acid Delivery: Choosing the Right Carriers,” Life 9(3):59 (2019), which are hereby incorporated by reference in their entirety) can be employed in the methods as described herein.

[0075] In alternative embodiments, the drug delivery vehicle is not linked per se to the conjugate, but the conjugate is encapsulated by, packaged within, or otherwise associated with the drug delivery vehicle. This is applicable to a number of the delivery vehicles identified above, including a nanoparticle delivery vehicle, a polymer-based particle, or a lipid-based particle.

[0076] Exemplary peptides or polypeptides suitable for cell uptake include, without limitation, an antibody or binding fragment thereof that binds a cell surface molecule, a cell penetrating peptide, or a toxin that facilitates cell uptake.

[0077] Suitable antibodies, or binding fragments thereof, that bind a cell surface molecule include, without limitation, those targeting a cell surface marker that is specific for a particular type of cell. Any number of cell specific surface markers are known in the art. In one embodiment, the cell specific surface marker is a tumor-specific antigen or cancer cell specific antigen, such as a cell surface protein expressed on oncogenic RAS cancer cells. Exemplary cancer cell specific surface markers (that the antibody, or binding fragment thereof, recognizes 171580388v1and specifically binds to) are selected from CUB domain-containing protein 1 (CDCP1), Intercellular adhesion molecule 1 (ICAM1), Integrin beta-5 (ITGB5), (5'-nucleotidase) NT5E, Tumor necrosis factor receptor superfamily member 3 (LTBR), Complement decay-accelerating factor (CD55), Aminopeptidase N (ANPEP), CD79, Trophoblast glycoprotein (TPBG), Integrin beta-1 (ITGB1), Prostaglandin F2 receptor negative regulator (PTGFRN), Integrin alpha-5 (ITGA5), and Exosome complex protein LRP1 (LRP1) (see e.g., Martinko et al., “Targeting RAS-driven Human Cancer Cells with Antibodies to Upregulated and Essential Cell-Surface Proteins,” eLIFE 7:e31098 (2018), which is hereby incorporated by reference in its entirety). Other cancer cell specific antigen can also be used for targeting by the antibody or binding fragment thereof.

[0078] Suitable cell penetrating peptides include, without limitation, TAT, cR10, Pep-1, penetratin, and transportan. Other cell penetrating peptides can also be used.

[0079] Suitable toxins that facilitate cell uptake are typically bacterial, fungal, or viral toxins, and in certain embodiments these toxins may be attenuated so that their toxic effects are diminished or abolished. Exemplary attenuated toxins include, without limitation, attenuated anthrax toxin, an attenuated diphtheria toxin, and attenuated Shiga or Shiga-like toxin. Other toxins, particularly attenuated toxins, can also be used.

[0080] Alternatively, any of a variety of non-proteinaceous delivery vehicles can be used, including a nanoparticle delivery vehicle, a polymer-based particle, or a lipid-based particle delivery vehicle.

[0081] Suitable nanoparticle delivery vehicles include, without limitation, gold nanoparticles, calcium phosphate nanoparticles, cadmium (quantum dots) nanoparticles, iron oxide nanoparticles, as well as particles derived from any other solid inorganic materials as known in the art.

[0082] Suitable polymer-based particles or polyplex carriers include cationic polymers such as polyethylenimine (PEI), and / or cationic polymers conjugated to neutral polymers, like polyethylene glycol (PEG) and cyclodextrin, as well as other proteins or polypeptides. Other suitable PEI conjugates to facilitate nucleic acid molecule or expression vector delivery in accordance with the methods described herein include, without limitation, PEI-salicylamide conjugates and PEI-steric acid conjugate. Other synthetic cationic polymers suitable for use as a delivery vehicle material include, without limitation, poly-L-lysine (PLL), polyacrylic acid (PAA), polyamideamine-epichlorohydrin (PAE) and poly[2-(dimethylamino)ethyl methacrylate] (PDMAEMA). Natural cationic polymers suitable for use as delivery vehicle material include, 171580388v1without limitation, chitosan, poly(lactic-co-glycolic acid) (PLGA), gelatin, dextran, cellulose, and cyclodextrin.

[0083] According to one embodiment, the carrier is a PEI-polypeptide conjugate that includes a protein or polypeptide carrier covalently bound to a plurality of PEI moieties, and this PEI-polypeptide conjugate is chemically linked to the mutant KRASG12Dbinding polypeptide or mutant KRASG12Dbinding peptide conjugate described above. As described in International PCT Application Publ. No. WO2022 / 224257, which is hereby incorporated by reference in its entirety, such PEI-polypeptide conjugates can use a number of polypeptide carriers, preferably though not exclusively plasma endogenous proteins such as albumin (e.g., human serum albumin or HSA), fibrinogen, and any of a variety of globulins, as well as designed ankyrin repeat proteins (DARPins).

[0084] In one example, the mutant KRASG12Dbinding polypeptide-E3 ligase subunit conjugate is linked to the PEI-albumin conjugate using a PEG linker to form conjugate that can deliver the KRASG12Dbinding polypeptide-E3 ligase subunit conjugate internally of the targeted cancer cells.

[0085] In another example, the mutant KRASG12Dbinding polypeptide-E3 ubiquitin ligase binding ligand conjugate is linked to the PEI-albumin conjugate using a PEG linker to form conjugate that can deliver the KRASG12Dbinding polypeptide-E3 ubiquitin ligase binding ligand conjugate internally of the targeted cancer cells.

[0086] In a further example, the mutant KRASG12Dbinding polypeptide-E2 ubiquitin- conjugating enzyme conjugate is linked to the PEI-albumin conjugate using a PEG linker to form conjugate that can deliver the KRASG12Dbinding polypeptide-E2 ubiquitin-conjugating enzyme conjugate internally of the targeted cancer cells.

[0087] Yet another example is the mutant KRASG12Dbinding polypeptide-E2 recruitment ligand linked to the PEI-albumin conjugate using a PEG linker to form conjugate that can deliver the KRASG12Dbinding polypeptide-E2 recruitment ligand conjugate internally of the targeted cancer cells.

[0088] Suitable lipid-based vehicles include, without limitation, cationic lipid based lipoplexes (e.g., 1,2-dioleoyl-3trimethylammonium-propane (DOTAP)), neutral lipids based lipoplexes (e.g., cholesterol and dioleoylphosphatidyl ethanolamine (DOPE)), anionic lipid based lipoplexes (e.g., cholesteryl hemisuccinate (CHEMS)), and pH-sensitive lipid lipoplexes (e.g., 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA)). Other suitable lipid-based delivery particles incorporate ionizable 171580388v1DOSPA in lipofectamine and DLin-MC3-DMA ((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31- tetraen-19-yl-4-(dimethylamino) butanoate).

[0089] Another aspect of the present disclosure is directed to polynucleotides encoding the mutant KRASG12Dbinding polypeptides or the mutant KRASG12Dbinding peptide conjugates described herein. The nucleic acid molecules of the present disclosure include isolated polynucleotides, portions of expression vectors or portions of linear DNA sequences, including linear DNA sequences used for in vitro transcription / translation, vectors compatible with prokaryotic, eukaryotic or filamentous phage expression, secretion and / or display of the compositions or directed mutagens thereof.

[0090] In one embodiment isolated polynucleotides of the present disclosure include those encoding the binding molecules described supra. Exemplary isolated polynucleotide molecules include those encoding the mutant KRASG12Dbinding polypeptide comprising one or more of an FG loop amino acid sequence of SEQ ID NO: 2, a BC loop amino acid sequence of SEQ ID NO: 13, and a DE loop amino acid sequence of SEQ ID NO: 7. In some embodiments, the FN domain encoded by the polynucleotide further comprises an amino acid substitution in one or more beta strands as discussed supra, including at one or more residues corresponding to residues Y31, R33, E47 and / or T49 of SEQ ID NO: 1 as well as any other substitutions described in Tables A and B. In some embodiments, the mutant KRASG12Dbinding polypeptide encoded by the polynucleotide of the disclosure comprises an amino acid sequence that is at least 80% identical to an amino acid sequence of SEQ ID NO: 18. In some embodiments, the mutant KRASG12Dbinding polypeptide encoded by the polynucleotide of the present disclosure comprises an amino acid sequence that is at least 90% identical to an amino acid sequence of SEQ ID NO: 18. In some embodiments, the polynucleotide of the present disclosure encodes the mutant KRASG12Dbinding polypeptide comprising an amino acid sequence of SEQ ID NO: 18 (MbKRAS_12D1).

[0091] In one embodiment isolated polynucleotides of the present disclosure include those encoding the binding molecules described supra. Exemplary isolated polynucleotide molecules include those encoding the mutant KRASG12Dbinding polypeptide comprising one or more of an FG loop amino acid sequence of SEQ ID NO: 3, a BC loop amino acid sequence of SEQ ID NO: 13, and a DE loop amino acid sequence of SEQ ID NO: 8. In some embodiments, the mutant KRASG12Dbinding polypeptide encoded by the polynucleotide further comprises an amino acid substitution in one or more beta strands as discussed supra, including at one or more residues corresponding to residues Y31, R33, E47 and / or T49 of SEQ ID NO: 1 as well as any other substitutions described in Tables A and B. In some embodiments, the mutant KRASG12D171580388v1binding polypeptide encoded by the polynucleotide of the disclosure comprises an amino acid sequence that is at least 80% identical to an amino acid sequence of SEQ ID NO: 19. In some embodiments, the mutant KRASG12Dbinding polypeptide encoded by the polynucleotide of the present disclosure comprises an amino acid sequence that is at least 90% identical to an amino acid sequence of SEQ ID NO: 19. In some embodiments, the polynucleotide of the present disclosure encodes the mutant KRASG12Dbinding polypeptide comprising an amino acid sequence of SEQ ID NO: 19 (MbKRAS_12D2).

[0092] In one embodiment isolated polynucleotides of the present disclosure include those encoding the binding molecules described supra. Exemplary isolated polynucleotide molecules include those encoding the mutant KRASG12Dbinding polypeptide comprising one or more of an FG loop amino acid sequence of SEQ ID NO: 4, a BC loop amino acid sequence of SEQ ID NO: 13, and a DE loop amino acid sequence of SEQ ID NO: 8. In some embodiments, the mutant KRASG12Dbinding polypeptide encoded by the polynucleotide further comprises an amino acid substitution in one or more beta strands as discussed supra, including at one or more residues corresponding to residues Y31, R33, E47 and / or T49 of SEQ ID NO: 1 as well as any other substitutions described in Tables A and B. In some embodiments, the mutant KRASG12Dbinding polypeptide encoded by the polynucleotide of the disclosure comprises an amino acid sequence that is at least 80% identical to an amino acid sequence of SEQ ID NO: 20. In some embodiments, the mutant KRASG12Dbinding polypeptide encoded by the polynucleotide of the present disclosure comprises an amino acid sequence that is at least 90% identical to an amino acid sequence of SEQ ID NO: 20. In some embodiments, the polynucleotide of the present disclosure encodes the mutant KRASG12Dbinding polypeptide comprising an amino acid sequence of SEQ ID NO: 20 (MbKRAS_12D3).

[0093] In one embodiment isolated polynucleotides of the present disclosure include those encoding the binding molecules described supra. Exemplary isolated polynucleotide molecules include those encoding the mutant KRASG12Dbinding polypeptide comprising one or more of an FG loop amino acid sequence of SEQ ID NO: 5, a BC loop amino acid sequence of SEQ ID NO: 13, and a DE loop amino acid sequence of SEQ ID NO: 8. In some embodiments, the mutant KRASG12Dbinding polypeptide encoded by the polynucleotide further comprises an amino acid substitution in one or more beta strands as discussed supra, including at one or more residues corresponding to residues Y31, R33, E47 and / or T49 of SEQ ID NO: 1 as well as any other substitutions described in Tables A and B. In some embodiments, the mutant KRASG12Dbinding polypeptide encoded by the polynucleotide of the disclosure comprises an amino acid sequence that is at least 80% identical to an amino acid sequence of SEQ ID NO: 21. In some 171580388v1embodiments, the mutant KRASG12Dbinding polypeptide encoded by the polynucleotide of the present disclosure comprises an amino acid sequence that is at least 90% identical to an amino acid sequence of SEQ ID NO: 21. In some embodiments, the polynucleotide of the present disclosure encodes the mutant KRASG12Dbinding polypeptide comprising an amino acid sequence of SEQ ID NO: 21 (MbKRAS_12D4).

[0094] In one embodiment isolated polynucleotides of the present disclosure include those encoding the binding molecules described supra. Exemplary isolated polynucleotide molecules include those encoding the mutant KRASG12Dbinding polypeptide comprising one or more of an FG loop amino acid sequence of SEQ ID NO: 5, a BC loop amino acid sequence of SEQ ID NO: 13, and a DE loop amino acid sequence of SEQ ID NO: 7. In some embodiments, the mutant KRASG12Dbinding polypeptide encoded by the polynucleotide further comprises an amino acid substitution in one or more beta strands as discussed supra, including at one or more residues corresponding to residues Y31, R33, E47 and / or T49 of SEQ ID NO: 1 as well as any other substitutions described in Tables A and B. In some embodiments, the mutant KRASG12Dbinding polypeptide encoded by the polynucleotide of the disclosure comprises an amino acid sequence that is at least 80% identical to an amino acid sequence of SEQ ID NO: 23. In some embodiments, the mutant KRASG12Dbinding polypeptide encoded by the polynucleotide of the present disclosure comprises an amino acid sequence that is at least 90% identical to an amino acid sequence of SEQ ID NO: 23. In some embodiments, the polynucleotide of the present disclosure encodes the mutant KRASG12Dbinding polypeptide comprising an amino acid sequence of SEQ ID NO: 23 (MbKRAS_12D5).

[0095] Another aspect of the disclosure is directed to a fusion construct, said fusion construct comprising a first portion, said first portion comprising polynucleotide encoding the mutant KRAS binding polypeptide as describe herein and a second portion operatively coupled to said first portion, said second portion comprising a polynucleotide encoding a E3 ubiquitin ligase subunit or an imaging protein or peptide thereof.

[0096] In any embodiment, the second portion of a KRAS fusion construct is a polynucleotide encoding an E3 ubiquitin ligase subunit. Suitable polynucleotides include those that encode an E3 ubiquitin ligase subunit selected from the group consisting of speckle type POZ protein (SPOP; Genbank Accession NP_003930), E3 ubiquitin-protein ligase CHIP (Genbank Accession NP_005852), Ankyrin repeat and SOCS box protein 1 (ASB1; Genbank Accession NP_001035535), Suppressor of cytokine signaling 2 (SOCS2; Genbank Accession NP_003868), DNA damage-binding protein 2 (DDB2; Genbank Accession NP_0000098), Protein cereblon (CRBN; Genbank Accession NP_057386), von Hippel-Lindau disease tumor 171580388v1suppressor (VHL; Genbank Accession NP_000542), S-phase kinase-associated protein 2 (SKP2; Genbank Accession NP_005974), F-box / WD repeat-containing protein 7 (FBW7; Genbank Accession NP_060785), F-box / WD repeat-containing protein 1A isoform 2 (Genbank Accession NP_003930) (see Lim et al., “bioPROTACs as Versatile Modulators of Intracellular Therapeutic Targets Including Proliferating Cell Nuclear Antigen (PCNA),” PNAS 117(11): 5791-5800 (2020), which is hereby incorporated by reference in its entirety. Each of the above-identified Genbank Accessions is also incorporated herein by reference in its entirety.

[0097] In any embodiment, the second portion of a KRAS fusion construct is a polynucleotide encoding an imaging agent. In any embodiment, the imaging agent is a fluorescent or luminescent proteins. Exemplary fluorescent proteins include, without limitation, Aequorea green fluorescent protein and derivatives thereof, Anthozoan fluorescent protein and derivatives thereof, Discosoma red fluorescent protein and derivatives thereof, Anemonia fluorescent protein and derivatives thereof. Exemplary luminescent proteins include, without limitation, Firefly luciferase protein and derivatives thereof, Renilla luciferase protein and derivatives thereof, and bacterial luciferase protein and derivatives thereof.

[0098] The polynucleotides of the disclosure may be produced by chemical synthesis such as solid phase polynucleotide synthesis on an automated polynucleotide synthesizer and assembled into complete single or double stranded molecules. Alternatively, the polynucleotides of the disclosure may be produced by other techniques such PCR followed by routine cloning. Techniques for producing or obtaining polynucleotides of a given known sequence are well known in the art.

[0099] The polynucleotides described herein may comprise at least one non-coding sequence, such as a promoter or enhancer sequence, intron, polyadenylation signal, a cis sequence facilitating RepA binding, and the like. The polynucleotide sequences may also comprise additional sequences encoding additional amino acids that encode for example a marker or a tag sequence such as a histidine tag or an HA tag to facilitate purification or detection of the protein, a signal sequence, a fusion protein partner such as RepA, Fc or bacteriophage coat protein such as pIX or pIII.

[0100] Another embodiment of the disclosure is a vector comprising at least one or more of the polynucleotides and fusion constructs as described herein. Such vectors may be plasmid vectors, viral vectors, vectors for baculovirus expression, transposon based vectors or any other vector suitable for introduction of the polynucleotides of the invention into a given organism or genetic background by any means. Such vectors may be expression vectors comprising nucleic acid sequence elements that can control, regulate, cause or permit expression of a polypeptide 171580388v1encoded by such a vector. Such elements may comprise transcriptional enhancer binding sites, RNA polymerase initiation sites, ribosome binding sites, and other sites that facilitate the expression of encoded polypeptides in a given expression system. Such expression systems may be cell-based, or cell-free systems well known in the art.

[0101] The polynucleotides as disclosed herein are preferably codon optimized for expression in mammalian cells, more preferably human cells. Methods of codon-optimization are known and have been described previously (e.g. International Application Publication No. WO 1996 / 09378 to Seed, which is hereby incorporated by reference in its entirety). A sequence is considered codon optimized if at least one non-preferred codon as compared to a wild-type sequence is replaced by a codon that is more preferred. Herein, a non-preferred codon is a codon that is used less frequently in an organism than another codon coding for the same amino acid, and a codon that is more preferred is a codon that is used more frequently in an organism than a non-preferred codon. The frequency of codon usage for a specific organism can be found in codon frequency tables that are well known and available in the art. Preferably more than one non-preferred codon, e.g. more than 10%, 40%, 60%, 80% of non-preferred codons, preferably most (e.g. at least 90%) or all non-preferred codons, are replaced by codons that are more preferred. Preferably the most frequently used codons in an organism are used in a codon- optimized sequence. Replacement by preferred codons generally leads to higher expression.

[0102] In any embodiment, the aforementioned polynucleotides are inserted into a vector, i.e., an expression vector for use as an in vivo therapeutic agent or for ex vivo production of the mutant KRASG12Dbinding polypeptide or conjugates described herein. Alternatively, these nucleic acid molecules may be inserted into an expression vector that is transformed or transfected into an appropriate host cell for expression and isolation of the encoded mutant KRASG12Dbinding polypeptide or conjugate.

[0103] In any embodiment, the vector comprising the polynucleotide encoding the mutant KRASG12Dbinding polypeptide or fusion construct is a viral vector. Suitable viral vectors include, without limitation, lentiviral vector, an adeno-associated viral vector, vaccinia vector, and a retroviral vector.

[0104] Another embodiment of the present disclosure is a host cell comprising the above described vectors. The mutant KRASG12Dbinding polypeptide and / or mutant KRAS binding peptide conjugates disclosed herein can be optionally produced by a cell line, a mixed cell line, an immortalized cell or clonal population of immortalized cells, as well known in the art (see e.g., Ausubel et al., eds., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, N.Y. (1987-2001); Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, 171580388v1Cold Spring Harbor, N.Y. (1989); Harlow and Lane, Antibodies, a Laboratory Manual, Cold Spring Harbor, N.Y. (1989); Colligan et al., eds., Current Protocols in Immunology, John Wiley & Sons, Inc., NY (1994-2001); Colligan et al., Current Protocols in Protein Science, John Wiley & Sons, NY, N.Y., (1997-2001), which are hereby incorporated by reference in their entirety).

[0105] The host cell chosen for expression may be of mammalian origin or may be selected from COS-1, COS-7, HEK293, BHK21, CHO, BSC-1, He G2, SP2 / 0, HeLa, myeloma, lymphoma, yeast, insect or plant cells, or any derivative, immortalized or transformed cell thereof. Alternatively, the host cell may be selected from a species or organism incapable of glycosylating polypeptides, e.g. a prokaryotic cell or organism, such as BL21, BL21(DE3), BL21-GOLD(DE3), XL1-Blue, JM109, HMS174, HMS174(DE3), and any of the natural or engineered E. coli spp., Klebsiella spp., or Pseudomonas spp. strains. Preferably, the host cell is a human cell line

[0106] In certain embodiments, the KRASG12Dbinding polypeptide, conjugate, or fusion protein of the present disclosure may be purified. As used herein, the term “purified” is intended to refer to a composition that is separable from other components, wherein the polypeptide, conjugate, or fusion protein is purified to any degree relative to its naturally available state. Thus, a purified polypeptide, conjugate, or fusion protein also refers to a polypeptide, conjugate, or fusion protein that is removed from its naturally occurring environment, i.e., when recombinantly produced, then removed from the host cell in which it was produced. When the term “substantially purified” is used, this means a composition in which the polypeptide, conjugate, or fusion protein forms the major component of the composition, such as the polypeptide, conjugate, or fusion protein constitutes about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of the composition.

[0107] Protein purification techniques are well known to those skilled in the art. At one level, these techniques involve a coarse fractionation of the cellular environment into polypeptide and non-polypeptide fractions. If the polypeptide has been isolated from other proteins, the polypeptide of interest may be further purified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity). Analytical methods particularly suitable for the preparation of pure peptides are ion exchange chromatography, exclusion chromatography; performing polyacrylamide gel electrophoresis; isoelectric focusing. Other methods of protein purification include ammonium sulfate precipitation, PEG, antibodies, etc. or by heat denaturation followed by centrifugation; gel filtration, reverse phase, hydroxyapatite and affinity chromatography; and combinations of these and other techniques. 171580388v1

[0108] In purifying polypeptides, conjugates, or fusion proteins of the present disclosure, it may be desirable to express the polypeptide, conjugate, or fusion protein in a prokaryotic or eukaryotic expression system and extract the polypeptide, conjugate, or fusion protein using denaturing conditions. The polypeptide, conjugate, or fusion protein may be purified from other cellular components using an affinity column that binds to a labeled portion of the polypeptide, conjugate, or fusion protein. As is generally known in the art, it is believed that the order in which the various purification steps are performed may be changed, or that certain steps may be omitted, and still result in a suitable method of preparing a substantially purified protein or peptide. The recovered polypeptide, conjugate, or fusion protein can be renatured as is known in the art.

[0109] The present disclosure provides pharmaceutical compositions comprising the KRASG12Dbinding polypeptide, conjugate, or fusion protein as disclosed herein. Such compositions comprise a prophylactically or therapeutically effective amount of the KRASG12Dbinding polypeptide, conjugate, or fusion protein and a pharmaceutically acceptable carrier. In a particular embodiment, the term “pharmaceutically acceptable” means approved by a federal regulatory agency or a state government or listed in the U.S. or European pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term “pharmaceutically acceptable carrier” refers to a diluent, excipient, and / or vehicle with which the therapeutic agent is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a particular carrier when the pharmaceutical composition is administered intravenously. Physiological saline solutions as well as aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Other suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.

[0110] The pharmaceutical compositions may also contain minor amounts of wetting or emulsifying agents or pH buffering agents, if desired. These compositions may also take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like. Oral formulations may include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. Examples of suitable pharmaceutical agents are described in "Remington's Pharmaceutical Sciences". Such compositions will contain a prophylactically or therapeutically effective amount of the KRASG12Dbinding polypeptide, 171580388v1conjugate, or fusion protein (preferably in purified form) and an appropriate amount of a carrier so as to provide a form for appropriate administration to a patient. The formulation should be suitable for the following modes of administration: oral, intravenous, intradermal, subcutaneous, intramuscular, intraperitoneal, intra-arterial, buccal, intranasal, nebulization, bronchial inhalation, and intratumoral injection.

[0111] Pharmaceutically acceptable salts include acid salts as well as salts with inorganic acids such as, for example, hydrochloric or phosphoric acids, or organic acids such as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as sodium, potassium, ammonium, calcium, or iron hydroxides, and organic bases such as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like.

[0112] In general, the ingredients of the compositions of the present disclosure are supplied separately or mixed together in unit dosage form, e.g., in the form of a dry lyophilized powder or an anhydrous concentrate in a hermetically sealed container such as an ampoule or sachet that indicates the amount of active agent. When the composition is to be administered by infusion, the composition can be dispensed in an infusion bottle containing sterile pharmaceutical grade water or saline. When the composition is administered by injection, an ampoule of sterile water or saline for injection may be provided so that the ingredients may be mixed prior to administration.

[0113] The compositions of the present disclosure may be formulated in a neutral form or in a salt form. Pharmaceutically acceptable salts include salts with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, and the like; and salts formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

[0114] Compositions of the type described above (e.g., containing the KRASG12Dbinding polypeptide) can also be used for diagnostic purposes.

[0115] As discussed below, the KRASG12D-binding polypeptides disclosed herein can be used for diagnostic purposes (i.e., screening for the presence of the KRAS(G12D)) and in small molecule screening assays to detect small molecules the exhibit high affinity binding to KRAS(G12D); and the KRASG12Dbinding polypeptide, conjugate, or fusion protein as disclosed for therapeutic purposes in treating KRAS(G12D)-mediated cancers. These are described more fully below.

[0116] KRAS(G12D) is associated with a number of cancers including, without limitation, malignant solid tumors, pancreatic adenocarcinoma, colon adenocarcinoma, lung 171580388v1adenocarcinoma (non-small lung cell and squamous cell carcinomas), colorectal adenocarcinoma, rectal adenocarcinoma, appendiceal carcinoma, breast carcinoma, gastric carcinoma, ovarian carcinoma, hepatocellular carcinoma, head and neck squamous cell carcinoma, glioblastoma, chordoma, thyroid carcinoma, and uveal melanoma. Detection of KRAS(G12D) as a marker for such cancers is therefore contemplated.

[0117] Due to their selectivity for KRAS(G12D), the KRASG12D-binding polypeptides disclosed herein can be used as diagnostic agents, including for in vitro diagnostic protocols (such as in various immunoassays where the KRASG12D-binding polypeptides replace antibodies / fragments thereof) and for in vivo diagnostic protocols (typically imaging procedures). Many suitable imaging agents are known in the art, as are methods for attaching them to antibodies or antibody-like molecules (see, e.g., U.S. Pat. Nos.5,021,236, 4,938,948, and 4,472,509, each of which is hereby incorporated by reference in its entirety). The imaging moiety used may be paramagnetic ions, radioisotopes, fluorescent dyes, NMR-detectable substances and X-ray imaging agents. In the case of the KRASG12D-binding polypeptides disclosed herein, attachment of the imaging moiety is preferably attached to the polypeptide at the N- or C-terminal end using well-known derivatization procedures so as not to disrupt the structure of the beta strands and loop formations as discussed above.

[0118] In general, the in vitro diagnostic binding methods comprise obtaining a sample suspected of containing a KRAS(G12D)-mediated cancer, and contacting the sample with a KRASG12D-binding polypeptide according to the present disclosure, as the case may be, under conditions effective to allow the formation of a binding complex. The term “sample”, as used herein, includes, but is not limited to, any quantity of a substance from a living thing or formerly living thing. Such living things include, but are not limited to, humans, mice, rats, monkeys, dogs, rabbits and other animals. Such substances include, but are not limited to, blood, (e.g., whole blood or components thereof), plasma, serum, urine, saliva, amniotic fluid, synovial fluid, endothelial cells, leukocytes, monocytes, other cells, organs, tissues, bone marrow, lymph nodes and spleen. By way of example, a tissue biopsy may be used as the sample and the tissue present in the biopsy sample can be subsequently treated to facilitate detection of KRAS(G12D), if present. Alternatively, the sample can be processed such that it is a cell-free sample and only contains soluble fractions from the originally obtained sample.

[0119] The KRASG12D-binding polypeptides will preferably be attached to a solid support (such as in the form of a column matrix, or a plate, or bead) and a sample suspected of containing KRAS(G12D)-mediated is applied to the immobilized KRASG12D-binding polypeptide. Unwanted components are washed from the solid support, leaving the 171580388v1KRAS(G12D) / KRASG12D-binding polypeptide complex, which are then collected from the solid support and counted.

[0120] The in vitro diagnostic binding methods also include methods for detecting and quantifying the amount of KRAS(G12D)-mediated cancer cells or associated components in a sample and the detection and quantification of any KRAS(G12D) / KRASG12D-binding polypeptide complex formed during the binding process. Here, a sample suspected of containing KRAS(G12D)-mediated cancer cells will be obtained and contacted with a KRASG12D-binding polypeptide that binds KRAS(G12D), followed by detection and quantification of the amount of such complexes formed under specified conditions. In terms of detection, the biological sample analyzed may be any sample suspected of containing a KRAS(G12D)-mediated cancer, such as a tissue section or specimen, a homogeneous tissue extract, a biological fluid (including blood and serum), or a secretion (such as stool or urine).

[0121] Contacting the selected biological sample with the KRASG12D-binding polypeptide under effective conditions for a period of time sufficient to allow complex formation is typically such an event: the composition containing the KRASG12D-binding polypeptide is simply added to the sample and the mixture is incubated for a period of time sufficient for the KRASG12D-binding polypeptide to form a complex (i.e., bind to KRAS)G12D)). After this time, the composition (such as a tissue section, ELISA plate, dot blot, or western blot) is typically washed to remove any non-specifically bound KRASG12D-binding polypeptide, allowing only those KRASG12D-binding polypeptide specifically bound within the primary complex to be detected.

[0122] In general, detection of complex formation is well known in the art and can be accomplished by applying a variety of methods. These methods are generally based on the detection of labels or markers, such as any of those radioactive, fluorescent, biological and enzymatic labels. Patents directed to the use of such labels include U.S. Pat. Nos.3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149, and 4,366,241, each of which is hereby incorporated by reference in its entirety. Of course, additional advantages may be found by using a secondary binding ligand (such as an antibody and / or biotin / avidin ligand) binding arrangement, as is known in the art.

[0123] The KRASG12D-binding polypeptide employed in the assay may itself be linked to a detectable label, wherein this label will then simply be detected, thereby allowing the amount of primary complex in the composition to be determined. Alternatively, a KRASG12D-binding polypeptide that becomes bound within the primary complex may be detected by a second binding agent ligand that has binding affinity for the KRASG12D-binding polypeptide. In these 171580388v1cases, the second binding ligand may be linked to a detectable label. The second binding partner is itself often an antibody and therefore may be referred to as a "secondary" antibody. Contacting the primary complex with a labeled secondary binding partner or antibody under effective conditions for a period of time sufficient to allow formation of a secondary complex. The secondary complexes are then typically washed to remove any non-specifically bound labeled secondary antibodies or ligands, and the label remaining in the secondary complexes is then detected.

[0124] Additional methods include detection of primary complexes by a two-step method. As described above, a secondary complex is formed using a second binding ligand (such as an antibody) having binding affinity for the KRASG12D-binding polypeptide. After washing, the secondary complexes are contacted again with a third binding ligand or antibody having binding affinity for the second antibody under effective conditions for a period of time sufficient to allow complexes (tertiary complexes) to form. A third ligand or antibody is linked to a detectable label, allowing the detection of the tertiary complexes thus formed. This system may provide signal amplification if desired.

[0125] In general, the in vivo diagnostic binding methods comprise introducing a pharmaceutical composition as disclosed herein where the KRASG12D-binding polypeptide includes a detectable label suitable for the specific form of diagnostic imaging. After administering the pharmaceutical composition to an individual, sufficient time is allowed for the KRASG12D-binding polypeptide to bind to KRAS(G12D) present in a tissue or population of cells. Thereafter, the individual is subjected to the imaging protocol and the location where specifically-bound KRASG12D-binding polypeptide is detected.

[0126] Imaging protocols include, without limitation, computed tomography, positron emission tomography, magnetic resonance imaging, and single-photon emission computed tomography.

[0127] Also contemplated are kits for use with the in vitro and in vivo diagnostic methods described above. Such kits include all necessary reagents, including the appropriately labeled (or unlabeled) KRASG12D-binding polypeptide(s) and any secondary ligands which may optionally be labeled. The kits will also include suitable containers and any solid supports, if appropriate, for in vitro diagnostics, and instructions for carrying out the diagnostic methods.

[0128] In addition, the KRASG12D-binding polypeptides disclosed herein can also be used as a reagent for establishing a competitive binding (displacement) assay for screening compounds that bind to KRAS(G12D). See, e.g., Quevedo et al., “Small Molecule Inhibitors of RAS-effector Protein Interactions Derived Using an Intracellular Antibody Fragment,” Nat 171580388v1Commun 9:3169 (2018), which is hereby incorporated by reference in its entirety. Competitive binding assays allow the high-throughput screening of combinatorial or natural compound libraries. This can be used to develop drugs that can bind to the same site on KRAS(G12D). Binding assays are also routinely used in finding and defining interaction partners and interactions linked with diseases.

[0129] Binding assays rely on the quantitative / qualitative detection of binding partners as well as the resulting binding complexes. For this purpose, either the target biomolecule, ligands or both need to be labelled. A plethora of labeling / detection techniques are used to perform binding assays including radioligand assays, affinity chromatography, surface plasmon resonance, isothermal titration calorimetry and light-based techniques using absorbance, fluorescence, or luminescence readouts. In addition, label-free, light-based techniques involving absorbance, reflectance, and / or interference can also be utilized.

[0130] A great number of binding assays, using microplate readers for detection of a light-based readout, have been described in the literature or are available in kit form. Light detection modes in common use are: absorbance, fluorescence, luminescence, Fluorescence Resonance Energy Transfer (FRET), Fluorescence polarization (FP), time-resolved fluorescence (TRF), TR-FRET (time-resolved fluorescence energy transfer), and AlphaScreen / AlphaLisa. In general, the techniques mentioned above can all be used in competitive binding assays for drug screening. Sensitivity, in a binding assay is commonly determined by calculating the limit of detection (LOD), defined as the minimum concentration of labelled ligand / complex that can be detected over the blank. Although the LOD value is very specific to each individual assay, general relative trends can be ascribed to each technique mentioned below.

[0131] Finally, given the role of KRAS(G12D) in the various types of cancer noted above, the disclosed KRASG12D-binding polypeptides, conjugates, and compositions, can be used to inhibit the mutant form of KRAS(G12D). See, e.g., Teng et al., “Selective and Noncovalent Targeting of RAS Mutants for Inhibition and Degradation,” Nat Commun 12:2656 (2021); Spencer-Smith et al., “Inhibition of RAS Function Through Targeting an Allosteric Regulatory Site,” Nat Chem Biol 13:62-68 (2017), each of which is hereby incorporated by reference in its entirety. The KRASG12D-binding polypeptides and conjugates themselves can be directly administered or, alternatively, polynucleotides and vectors (e.g. plasmid, virus, mRNA) encoding the same can be administered. See, e.g., Cronican et al., “Potent Delivery of Functional Proteins into Mammalian Cells in vitro and in vivo Using a Supercharged Protein,” ACS Chem Biol 5:747-752 (2010); Peraro & Kritzer, “Emerging Methods and Design Principles 171580388v1for Cell-Penetrant Peptides,” Angew Chem Int Ed Engl 57:11868-11881 (2018), each of which is hereby incorporated by reference in its entirety).

[0132] Effective amounts of the disclosed KRASG12D-binding polypeptides, conjugates, and compositions will depend on the nature of use, including the nature of the cancerous condition which is being treated, tumor volume and stage, and its location(s). By way of example only, suitable concentrations of the active agent may range from about 1 µM to about 10 mM, preferably about 10 µM to about 5 mM, about 50 µM to about 2 mM, or about 100 µM to about 1 mM. The volume of the composition administered, and thus, dosage of the peptide administered can be adjusted by one of skill in the art to achieve optimized results. By way of example, about 250 µg to about 2000 mg (such as about 500 µg to about 1000 mg; or about 1000 µg to about 500 mg) can be administered per day, repeated periodically as needed, e.g., every third day, once weekly, every other week, etc. This can be adjusted lower to identify the minimal effective dose, or tailored higher or lower according to the nature of the tumor(s) to be treated.

[0133] It is also contemplated that the above-identified active agents can be used alone or in combination with other surgical or non-surgical therapies to treat the cancerous condition. Exemplary non-surgical therapies include, without limitation, a chemotherapeutic agent, a radiation therapy, an antiangiogenic agent, an immunomodulating agent, and combinations thereof.

[0134] By “treating”, it is contemplated that in certain embodiments the growth rate of the cancer tumors can be slowed such that the disease state can be managed. Alternatively, it is also contemplated that in certain embodiments the growth of tumors can be halted or, more preferably, reduced in tumor volume. Such reductions can include the reduction in tumor volume such that surgical procedures in combination with the active ingredient are capable of eliminating the tumor. Alternatively, such reductions can include elimination of the tumor by the active ingredient alone or in combination with another non-surgical therapy (i.e., as a combination therapy). EXAMPLES

[0135] The examples below are intended to exemplify the practice of embodiments of the disclosure but are by no means intended to limit the scope thereof. Materials and Methods for Example 1-7

[0136] Protein preparation, monobody development, biophysical characterization, and crystal structure determination was performed essentially as described previously (Teng et al., 171580388v1“Selective and Noncovalent Targeting of RAS Mutants for Inhibition and Degradation,” Nat Commun 12:2656 (2021); Teng et al., “Engineering Binders with Exceptional Selectivity,” Methods Mol Biol 2491:143-154 (2022), each of which is hereby incorporated by reference in its entirety). Deep mutational scanning was performed following published methods (Fowler, “High-resolution Mapping of Protein Sequence-function Relationships,” Nat Methods 7:741-746 (2010); Hattori et al., “Creating MHC-Restricted Neoantigens with Covalent Inhibitors That Can Be Targeted by Immune Therapy,” Cancer Discov 13:132-145 (2023), each of which is hereby incorporated by reference in its entirety). Colocalization, immunoprecipitation, signaling assays, and mouse xenograft experiments were performed essentially as described previously (Khan et al., “Identification of the Nucleotide-free State as a Therapeutic Vulnerability for Inhibition of Selected ONCOGENIC RAS mutants,” Cell Rep 38:110322 (2022); Khan et al., Targeting the alpha4-alpha5 Dimerization Interface of K-RAS Inhibits Tumor Formation in vivo,” Oncogene 38:2984-2993 (2019), each of which is hereby incorporated by reference in its entirety).

[0137] Protein expression and purification: All proteins used for monobody development and binding assays, including RAS constructs (KRAS4B residues 1–174 containing G12C, G12V, G12D, G13D, WT) and monobodies, were produced with an N-terminal tag containing His6, Avi-tag for biotinylation and a TEV protease recognition site using the pHBT vector. The RAS constructs were constructed by Kunkel mutagenesis using a vector encoding the wild-type RAS as the template (Teng et al., “Selective and Noncovalent Targeting of RAS Mutants for Inhibition and Degradation,” Nat Commun 12:2656 (2021), which is hereby incorporated by reference in its entirety). Monobody genes were amplified from yeast cells and cloned into the pHBT vector (Sha et al., “Dissection of the BCR-ABL Signaling Network Using Highly Specific Monobody Inhibitors to the SHP2 SH2 Domains,” Proc Natl Acad Sci USA 110:14924-14929 (2013), which is hereby incorporated by reference in its entirety). The proteins were produced in E. coli BL21 (DE3). To produce biotinylated proteins, E. coli BL21(DE3) with the pBirA plasmid was used as the host and grown in the presence of 50 mM of biotin. Expressed proteins were purified using Ni-Sepharose columns (Cytiva) via gravitational flow, followed by dialysis in Tris-buffered saline (TBS, 50 mM Tris-Cl pH 7.5 containing 150 mM NaCl) for non-RAS proteins. TBS containing 20 mM MgCl2and 0.5 mM DTT was used for RAS proteins. Samples were further purified using a Superdex 75 size exclusion column on an AKTA Pure system (Cytiva). The Cys-light version (C51S;C80L;C118S) (Ostrem et al., “K- Ras(G12C) Inhibitors Allosterically Control GTP Affinity and Effector Interactions,” Nature 503:548-551 (2013), which is hereby incorporated by reference in its entirety) of KRAS(G12D) (residues 1–169) and monobodies used for crystallization and BLI were produced with the pHBT 171580388v1vector in E. coli BL21(DE3) as described above. Following Ni-Sepharose purification, the N- terminal His6and Avi tags were removed with TEV protease in the presence of 0.5 mM EDTA and 0.5 mM DTT during overnight dialysis against TEV cleavage buffer (50 mM Tris-Cl pH 8, 50 mM NaCl, 5 mM β-mercaptoethanol, supplemented with 20 mM MgCl2for KRAS). Cleaved tags and TEV proteases were removed by Ni-Sepharose, followed by size-exclusion chromatography.

[0138] Nucleotide exchange of RAS: Purified RAS proteins used in binding experiments and crystallization were prepared by diluting stock protein (typically containing 20–250 mM RAS) 25 times with 20 mM Tris-Cl buffer pH 7.5 containing 5 mM EDTA, 0.1 mM DTT, and 1 mM final concentration of a nucleotide (GDP or GTPγS). Samples were incubated at 30 °C for 30 min. MgCl2was then added to the sample at a final concentration of 20 mM and the solution was further incubated on ice for at least 5 minutes prior to use.

[0139] Monobody development: General procedures for the development of monobodies against purified protein targets have been described previously (Teng et al., “Selective and Noncovalent Targeting of RAS Mutants for Inhibition and Degradation,” Nat Commun 12:2656 (2021); Koide et al., “Teaching an Old Scaffold New Tricks: Monobodies Constructed Using Alternative Surfaces of the FN3 Scaffold,” J Mol Biol 415:393-405 (2012), each of which is hereby incorporated by reference in its entirety). After four rounds of phage display library selection using biotinylated KRAS(G12D) at concentrations of 100, 100, 50, and 20 nM, the genes encoding monobodies from the enriched phage pool were transferred to a yeast display vector, which was used to construct yeast-display libraries. The yeast display libraries were sorted using a S3e fluorescence-activated cell sorter (Bio-Rad). The first round of sorting recovered clones that bound to KRAS(G12D); the second round recovered clones that did not bind to KRAS(WT); and the third round recovered clones that bound to KRAS(G12D). Single clones were then screened for selective binding to RAS mutants. The expression of monobodies on the surface of yeast cells was detected using mouse anti-V5 (ThermoFisher, MA5-15253, 1:75 for sorting approximately 107yeast cells, and 1:300 for staining 105yeast cells for analysis) followed by labeling using anti-mouse IgG-FITC conjugate (Millipore Sigma, F0257, 1:100). Target binding was detected with NeutrAvidin Dylight650 (Invitrogen). Yeast cells were analyzed using an iQue flow cytometer (Sartorius). The median of the signal intensity in the Dylight650 channel for the 75–95th percentile population was taken as a representative signal. This sampling method of flow cytometry events minimizes erroneous contributions from events with anomalously high signals while retaining events with high signals. 171580388v1

[0140] Biolayer interferometry (BLI) analysis: BLI experiments were performed on an Octet Red96e instrument (Sartorius). Biotinylated KRAS proteins bound to nucleotide were immobilized on streptavidin biosensor tips. Samples were diluted in 20 mM HEPES-NaOH buffer (pH 7.4) containing 150 mM NaCl, 5 mM MgCl2, 0.2 mM TCEP and 0.005% Tween-20. BLI signals were analyzed using Octet Data Analysis software (Sartorius).

[0141] Crystallization and X-ray structure determination: Purified and tag-cleaved Cys- light KRAS(G12D) bound to GTPγS or GDP was incubated with purified, tag-cleaved monobody 12D1(K63S) or 12D5(K63S) at a 1:1.1 molar ratio. The K63S mutation was introduced to facilitate crystallization (Wojcik et al., “A Potent and Highly Specific FN3 Monobody Inhibitor of the Abl SH2 Domain,” Nat Struct Mol Biol 17:519-527 (2010), which is hereby incorporated by reference in its entirety). The complexes were purified with a Superdex 7510 / 300 SEC column (Cytiva) in 10 mM NaCl, 10 mM Tris-Cl pH8, 20 mM MgCl2and concentrated to approximately 10 mg / ml. The 12D1(K63S)-KRAS(G12D)•GTPγS complex was crystallized in 2.8 M sodium acetate trihydrate at pH 7.0 when mixed 1:1 in a total volume of 2 ml using a sitting drop vapor diffusion method. The 12D5(K63S)-KRAS(G12D)•GTPγS complex was crystalized in 0.2 M sodium sulfate decahydrate containing 20% w / v PEG 3350 and 0.05% w / v benzamidine hydrochloride when mixed 1:1 in a total volume of 2 ml using a sitting drop vapor diffusion method. Crystals were preserved in the reservoir solution with an additional 20% (v / v) glucose. X-ray diffraction data were collected at the Advance Photon Source at the Argonne National Laboratory using beam line 19-ID. Diffraction data were processed using HKL3000 and HKL2000 (Minor et al., “HKL-3000: The Integration of Data Reduction and Structure Solution--From Diffraction Images to an Initial Model in Minutes,” Acta Crystallogr D Biol Crystallogr 62:859-866 (2006); Otwinowski & Minor, “Processing of X-ray Diffraction Data Collected in Oscillation Mode,” Methods Enzymol 276:307-326 (1997), each of which is hereby incorporated by reference in its entirety). The starting models were built by molecular replacement using PDB entries 5VPZ, 5VP7 and 7L0G as search models using Phaser (McCoy et al., “Phaser Crystallographic Software,” J Appl Crystallogr 40:658-674 (2007), which is hereby incorporated by reference in its entirety). Structural refinement was performed iteratively by using phenix.refine (Afonine et al., “Towards Automated Crystallographic Structure Refinement with Phenix.refine,” Acta Crystallogr D Biol Crystallogr 68:352-367 (2012), which is hereby incorporated by reference in its entirety), Coot (Emsley et al., “Features and Development of Coot,” Acta Crystallogr D Biol Crystallogr 66:486-501 (2010), which is hereby incorporated by reference in its entirety), and PDBredo (Joosten et al., “The PDB_REDO Server for Macromolecular Structure Model Optimization,” IUCrJ 1:213-220 171580388v1(2014), which is hereby incorporated by reference in its entirety). Protein-protein interaction interfaces were analyzed by using PDBePISA server available at the website for the European Bioinformatics Institute of the European Molecular Biology Laboratory (see Krissinel & Henrick, “Inference of Macromolecular Assemblies from Crystalline State,” J Mol Biol 372:774- 797 (2007), which is hereby incorporated by reference in its entirety). The root-mean-square deviations (RSMDs) were calculated by using the Structure Pairwise Alignment Tool available at the Research Collaboratory for Structural Bioinformatics, Protein Data Bank (“RCSB PDB”) website.

[0142] Deep mutational scanning: A yeast-display library of 12D4 was constructed by changing residues V27, T28, V29, V30, F31, D33, E47, T49, S51, G52, S53, K75, Y76, L77, F78, W79, S80, G81 and Y82 to all 20 amino acids, one amino acid at a time. Oligonucleotides including the NNK codon, where N is a mixture of A, T, G and C and K is a mixture of G and T, for the intended randomized position were used to introduce the mutations using PCR. Three separate reactions were performed with pooled oligonucleotides for closely located positions: sub-library 1 for V27, T28, V29, V30, F31 and D33; sub-library 2 for E47, T49, S51, G52 and S53; and sub-library 3 for K75, Y76, L77, F78, W79, S80, G81 and Y82. Yeast display vectors encoding the full-length monobody genes containing mutations fused with aga2 gene and V5 tag were constructed using yeast homologous recombination. Next, the yeast cells containing the combined library were incubated with either biotinylated KRAS G12D complexed with GTPγS or GDP, either 10 nM or 100 nM, as well as anti-V5 antibody, using the conditions described in the monobody development section above, and the binding and non-binding populations were sorted. Plasmids encoding the monobody genes were purified using Zymoprep Yeast Plasmid Miniprep II (Zymo Research Corporation, D2004), and the monobody genes were amplified using primers that are designed for amplicon sequencing with different index sequences. The PCR products were sequenced using MiSeq (Illumina). Sequencing data were analyzed using a set of UNIX and Python scripts developed in house to deduce the number of reads for each mutation.

[0143] Cell culture: Cell lines (HEK293T, HPAF-II) used in the study were directly purchased from ATCC, H358 was validated externally via IDEXX (H358), Panc-1 cells were previously validated by STR analysis (Khan et al., “Identification of the Nucleotide-free State as a Therapeutic Vulnerability for Inhibition of Selected Oncogenic RAS Mutants,” Cell Rep 38:110322 (2022), which is hereby incorporated by reference in its entirety). Pa14C and PSN1 cells were the kind gifts of Dr. Aaron Hobbs and were previously validated by STTR analysis. HEK293T, Panc-1 and HPAF-II cells were maintained in DMEM high glucose with L-glutamine 171580388v1(Hyclone) supplemented with 10% FBS (Gemini Bio-products) and antibiotics-antimycotics (Gibco). H358 cells were maintained in RPMI-1640 high glucose with sodium pyruvate, L- glutamine (Thermo), supplemented with 10% FBS and penicillin / streptomycin. The absence of mycoplasma contamination was periodically confirmed using a PCR-based mycoplasma testing kit (eMyco, Bulldog Bio). Pa14C and PSN1 cells were grown in RPMI with 10% FBS. All culture media were purchased commercially (Corning). Transient transfection in HEK-293, HEK-293T and NIH / 3T3 cells was done using polyethyleneimine (PEI). For HEK-293 and HEK-293T, typically, 3 ml of 1 mg / ml PEI stock was used for each mg of DNA in Opti-MEM reduced serum media (Life Technologies). Opti-MEM-PEI mixture was incubated for 10 minutes at room temperature. Next, DNA was added to Opti-MEM: PEI cocktail and incubated for at least 20 minutes at room temperature. The Opti-MEM: PEI-DNA mixture was added to cells in serum-free media and incubated for three hours after which the media was replaced with fresh complete media. Transfections in NIH / 3T3 cells were done with the same procedures, however, 5 ml of 1 mg / ml PEI stock was used for each mg of DNA.

[0144] Colocalization assays: HEK293T cells were cultured in glass-bottom 8-well chambers (ibidi GmbH) for colocalization assays for 1 day prior to transfection. On the day of transfection at 70–90% confluency, media were replaced with antibiotics-free complete media (DMEM supplemented with 10% FBS). The vectors encoding mCherry-fused monobodies were constructed by cloning their genes into the pEGFP vector at the NheI and ApAI restriction enzyme sites. The pEGFP KRAS4B vector was a gift of Prof. Mark Philips. Transfection of HEK293T cells with pEGFP vectors encoding the appropriate mCherry fused monobodies and EGFP fused KRAS4B constructs was performed with lipofectamine 3000 (Thermo Fisher Scientific) and according to the manufacturer’s recommended protocol. On the following day, transfected cells were imaged with a LSM710 confocal microscope (Zeiss).

[0145] Pull-down assays: HPAF-II, Panc1 and H358 cells were cultured in 10 cm plates (Corning, #430167). Cells were lysed by incubating them on ice for 15 min in GTPase lysis buffer (25 mM Tris-Cl pH 7.2, 150 mM NaCl, 5 mM MgCl2, 1% NP-40, and 5% glycerol supplemented with protease inhibitor tablet (Roche, 5892991001) and phosphatase inhibitors (1 mM sodium orthovanadate, 10 mM NAF, 54 mM β-glycerol phosphate)) immediately before analysis. After centrifugation for 15 min at 15,000 × g, the supernatants were collected and incubated with SA agarose resins (Thermo Fisher Scientific) for 1h at 4 °C to remove molecules that nonspecifically bound to the resins. After the removal of the resins via centrifugation, the precleared lysates were then incubated with biotinylated monobodies or the RAS-Binding domain (RBD) of RAF bound to SA agarose resins for 3 hr at 4 °C while rotating. The agarose 171580388v1resins were then washed twice with the GTPase lysis buffer and boiled in 1x SDS buffer with 37.5 mM b-mercaptoethanol and processed for western blotting. RAS proteins were detected using a mouse pan-RAS antibody (SCBT, sc-166691, 1:500) followed by anti-mouse HRP conjugate (Pierce, 31432, 1:4000) with the ECL 2 Western blotting substrate (Pierce; 80196). Example 1 - Development of the Initial Monobody Selective to KRAS(G12D), 12D1

[0146] To develop monobodies that demonstrate high selectivity for KRAS(G12D), established methods were used that combine phage display and yeast display technologies. Initial selection was performed using naïve phage-display libraries with KRAS(G12D)•GTPγS as the target for positive selection and KRAS(WT)•GDP for negative selection. Throughout this work, the KRAS4B isoform was used exclusively; KRAS4B is simply referred to hereinafter as KRAS for brevity. After rounds of phage-display library sorting, the enriched pool of monobody clones was transferred to a yeast display library. Using fluorescence-activated cell sorting (FACS), clones were screened for binding the KRAS(G12D)•GTPγS and negligibly binding to KRAS(WT)•GDP or KRAS(WT)•GTPγS. Clone 12D1 was identified as showing high selectivity towards KRAS(G12D)•GTPγS, weaker binding to KRAS(G12D)•GDP and negligible binding to KRAS(WT) and other KRAS mutants tested in either GDP or GTPγS bound state (FIGS.1A-1B). Bio-layer interferometry (BLI) using purified protein samples further demonstrated that 12D1 bound at least 25-fold more tightly to KRAS(G12D)•GTPγS than to KRAS(WT)•GTPγS and KRAS(WT)•GDP (FIG.1C). This monobody bound to KRAS(G12D)•GTPγS ~9-fold more tightly than to KRAS(G12D)•GDP (FIG.1C). It inhibited the binding of RAF RBD to KRAS(G12D)•GTPγS with an IC50value of 54 nM (FIG.1D), indicating that the interaction of KRAS(G12D) with RAF RBD and 12D1 was mutually exclusive. These results demonstrate the feasibility of developing monobody inhibitors highly selective to KRAS(G12D).

[0147] 12D1 did not bind to HRAS(G12D)•GTPγS (FIG.1E) or to NRAS(G12D)•GTPγS (FIG.1F). Residue 95, located in the S-II pocket, is not conserved among the RAS isoforms. It is Gln in HRAS and Leu in NRAS. The introduction of the Q95H mutation in HRAS was found to mimic KRAS, which promoted the binding of 12D1 to HRAS bearing Q95H (Fig.1e). This indicates that 12D1 specifically recognizes three features of KRAS(G12D), i.e., the G12D mutation, the GTP-bound state, and His95. A KRAS(G12C) inhibitor, sotorasib (AMG510), similarly recognizes H95, one of three residues that form a pocket for drug binding (Lanman et al., “Discovery of a Covalent Inhibitor of KRAS(G12C) 171580388v1(AMG 510) for the Treatment of Solid Tumors,” J Med Chem 63:52-65 (2020), which is hereby incorporated by reference in its entirety). Example 2 - Structural Basis for the Selectivity of 12D1 Towards KRAS(G12D)

[0148] To elucidate the mechanism underlying the specific recognition of KRAS(G12D) by monobody 12D1, a surface mutant version of 12D1, 12D1(K63S), was crystallized in complex with the “Cys-light” version of KRAS(G12D), and the structure was determined at 2.52 Å (Table 2, infra). For brevity, 12D1(K63S) is referred to in the remainder of Example 2 as 12D1 and the Cys-light version of KRAS is referred to as KRAS. None of these mutations are located near the monobody-KRAS interface.

[0149] The monobody and KRAS molecules retained their overall folds, as expected (FIG.2A). The monobody framework excluding the diversified regions takes on a conformation close to the parental FN3 domain structure (PBD: 1FNA, RMSD for Cα = 0.63 Å). Similarly, the RAS molecule excluding the switch regions and the nucleotide is similar to a previous KRAS(G12D)•GppNHp structure (PDB: 5USJ, RMSD for Cα = 0.69 Å). The interface area between the two molecules is 914 Å2, a typical interface size for monobody–target complexes (Gilbreth & Koide, “Structural Insights for Engineering Binding Proteins Based on Non- antibody Scaffolds,” Curr Opin Struct Biol 22:413-420 (2012), which is hereby incorporated by reference in its entirety). Interestingly, it was found that the nucleotide in the crystal was more consistent with GDP than GTPγS that was utilized for crystallization (FIGS.7A-7B). It is believed that GTPgS was slowly hydrolyzed to GDP during the crystallization process, as reported previously for other GTPases (O'Donnell et al. “Timing and Reset Mechanism of GTP Hydrolysis-Driven Conformational Changes of Atlastin,” Structure 25:997-1010 e1014 (2017); Berghuis et al., “Structure of the GDP-Pi Complex of Gly203--> Ala galpha1: A Mimic of the Ternary Product Complex of γalpha-catalyzed GTP Hydrolysis,” Structure 4:1277-1290 (1996), each of which is hereby incorporated by reference in its entirety). The affinity of 12D1 to KRAS(G12D)•GDP is high enough to maintain the complex at high protein concentrations used for crystallization. Thus, the structure represents the complex of monobody 12D1 and KRAS(G12D)•GDP. The structure of an affinity-matured monobody bound to KRAS(G12D)•GTPγS is described in Example 5.

[0150] The crystal structure revealed that 12D1 bound primarily to the S-II pocket, the cleft between the switch II and the α3 helix (FIGS.2B, 7C). This binding surface differs from another RAS mutant-specific monobody, 12VC1, that binds to both switch I and II regions and 171580388v1selectively recognizes the G12C and G12V mutants (Teng et al., “Selective and Noncovalent Targeting of RAS Mutants for Inhibition and Degradation,” Nat Commun 12:2656 (2021), which is hereby incorporated by reference in its entirety) (FIG.7D).12D1 mainly interacts with the P- loop and the switch II region via the FG loop (residues 78-82), and with the α3 helix via the C and D stands and the DE-loop (FIGS.7C, 7D). This mode of recognition of the S-II pocket is similar to other inhibitors specific to KRAS(G12D), i.e., the KD2 peptide, the KRpep-2d peptide and MRTX1133 (Sakamoto et al. “Generation of KS-58 as the First K-Ras(G12D)-inhibitory Peptide Presenting Anti-cancer Activity in vivo,” Sci Rep 10:21671 (2020); Zhang et al., “GTP- State-Selective Cyclic Peptide Ligands of K-Ras(G12D) Block Its Interaction with Raf,” ACS Cent Sci 6:1753-1761 (2020); Wang et al., “Identification of MRTX1133, a Noncovalent, Potent, and Selective KRAS(G12D) Inhibitor,” J Med Chem 65:3123-3133 (2022), each of which is hereby incorporated by reference in its entirety) (FIGS.2A-2B), confirming that S-II pocket is a “druggable site” for achieving selectivity to KRAS(G12D).

[0151] Although 12D1 and the other three inhibitors of KRAS(G12D) bind to the S-II pocket, 12D1 presents substantially more bulk in the pocket (FIG.2C). W78, Y79, S80, G81 and Y82 of 12D1 occupy the space that is occupied by residues of the RAS switch II region in the other structures, and consequently capture an enlarged SII pocket conformation (see Example 3).

[0152] Closer inspection of the interface revealed that 12D1 directly interacts with the side chain of Asp12RASusing a hydrogen bond with the backbone NH group of Gly81Mb(FIGS. 2B, 7C). For clarity, hereinafter residue numbers for RAS and 12D1 will be indicated with RAS and Mb in superscript, respectively. This interaction bears striking resemblance with that between the nitrogen atom of MRTX1133 and the Asp12RASside chain (FIG.2B) (Wang et al., “Identification of MRTX1133, a Noncovalent, Potent, and Selective KRAS(G12D) Inhibitor,” J Med Chem 65:3123-3133 (2022), which is hereby incorporated by reference in its entirety). In contrast, other KRAS(G12D)-directed peptide inhibitors with moderate selectivity do not form direct contacts with the Asp12RASside chain (Fig.2B, the right two panels). Taken together, the observed direct interactions with the Asp12RASside chain rationalize the high selectivity of 12D1 and MRTX1133 to the G12D mutant.

[0153] In addition to Gly81Mb, the backbone of Ser80Mbforms multiple hydrogen bonds with Ala59RASand Gln61RAS, indicating that Ser80Mbis another key residue for the interaction between 12D1 and KRAS(G12D) (FIGS.2B, 7C). The side chain of Thr49Mband Phe31Mbinteract with the side chain of His95RASin the α3 helix via a hydrogen bond and π−π stacking interaction (FIG.7E), respectively, indicating its contribution to selective binding of 12D1 to 171580388v1KRAS(G12D) over HRAS(G12D) and NRAS(G12D) (FIG.1E). These structural features rationalize the specific recognition of KRAS(G12D) by 12D1. Example 3 - 12D1 Captures an Enlarged S-II Pocket Conformation

[0154] Similar to other G12D-selective inhibitors reported to date, 12D1 binds to the switch II pocket. However, the conformations of the switch I and II regions of KRAS(G12D) in the 12D1 complex are substantially different from those of the regions in previously reported RAS structures (FIG.8A). The greater volume of 12D1 moieties that occupy the S-II pocket displaces switch II toward switch I. The loop region between the β3 strand and the α2 helix, including the switch II region, in the 12D1-bound KRAS(G12D) is shifted toward the switch I region (“up” in the depicted figure) compared with the region in other RAS structures (FIGS. 2D, 8B). Switch I region in the 12D1–KRAS(G12D) complex also takes on a conformation distinct from that in KRAS(G12D) bound to other inhibitors as well as in RAS structures in the absence of a bound inhibitor (FIGS.2D, 8A). Unfortunately, factors contributing to the stabilization of the observed conformation of switch I cannot be unambiguously defined, because the switch I region is involved in crystal packing (FIG.8C). These differences are likely to be caused by 12D1 presenting larger bulk in the S-II pocket. Consistent with this view, the pocket formed between switch II loop and the α3 helix is substantially larger in the 12D1 complex than the other complexes with G12D inhibitors (FIG.2E). To quantify these changes, the distance between the Cα positions of E62 in switch II and Q99 in α3 in each structure was measured (FIG.2E). The distance in the 12D1 complex (22.4 Å) is much greater than that in the MRTX1133 complex (12.0 Å) and the cyclic peptide complexes (16.4 and 14.0 Å, respectively).

[0155] Together, this structural analysis revealed yet another conformation of the switch I and II regions of RAS(G12D), further defining the plasticity of the switch regions in this RAS mutant. It also revealed the most open form of the switch II groove to date, which may be useful for structure-guided drug discovery. Example 4 - Affinity Maturation of 12D1 Monobody

[0156] To improve the potency of 12D1 affinity, its gene was subjected to error-prone PCR and the resulting mutants were screened for higher affinity clones, which yielded 12D2 that contained two mutations, I20V and P51S, both located outside the diversified positions in the starting library (FIG.3A).12D2 had approximately four-fold higher affinity to KRAS(G12D)•GTPγS than 12D1 and also showed less discrimination of the GTPgS- and GDP- 171580388v1bound states (FIGS.3B, 9A). In the crystal structure, P51Mbis located near the interface with RAS(G12D), indicating that the P51S mutation improved the binding interface. Ile20Mbis located away from the interface and its side chain is a part of the hydrophobic core of the monobody. As such, the role of the I20V mutation is unclear.

[0157] Next, directed mutations were introduced to residues in the FG loop of 12D2, the segment containing most residues that form extensive contacts with KRAS(G12D) in the crystal structure of 12D1 (Figs.2A-2E). Three adjacent randomized positions were introduced in the 9- residue sequence of the FG loop, KYLWYSGYS (resides 1-9 of SEQ ID NO: 2; FIG.3A), to generate three sub-libraries (XXXWYSGYS, SEQ ID NO: 14; KYLXXXGYS, SEQ ID NO: 15; and KYLWYSXXX, SEQ ID NO: 16, where in each instance X denotes a randomized position with all 20 genetically encoded amino acids). This was followed by library sorting with yeast display. These experiments resulted in clones 12D3 and 12D4 having further improved affinity to KRAS(G12D)•GTPγS (FIGS.3C, 3D, 9D, 9G).12D4 contained two mutations W78F and Y79W, and 12D3 contained an additional mutation, S80K (Fig.3A), originating from the library in which the middle three residues (positions 78-80) were randomized.12D3 maintained the preference toward KRAS(G12D)•GTPγS over KRAS(G12D)•GDP (Fig.3C) as seen in 12D1 and 12D2. In contrast, 12D4 bound to both GTPγS- and GDP-bound forms of KRAS(G12D) with similar affinity (Fig.3D). These monobodies exhibited high selectivity for KRAS(G12D), with up to a 180-fold difference in affinity between KRAS(G12D) and KRAS(WT) (FIGS.3B, 3C, 3D). All the monobodies bound only very weakly to other KRAS mutants in either nucleotide-bound form as tested in the yeast display format (FIGS.9B, 9E, 9H), and discriminated KRAS(G12D) and HRAS(G12D) by recognizing the difference at position 95 (FIGS.9C, 9F, 9I). A pull-down assay with clone 12D2 further confirmed its selectivity towards the KRAS(G12D) mutant.12D2 selectively captured endogenous RAS from cancer cell lines Panc-1 and HPAF-II that contain KRAS(G12D), but not from H358 cells containing KRAS(G12C) (FIG.3E). Together, the affinity of the initial monobody was successfully improved while maintaining its high selectivity to KRAS(G12D). Example 5 - Crystal Structures of 12D5 Monobody in Complex with KRAS(G12D)•GTPγS and with KRAS(G12D)•GDP

[0158] Attempts to crystallize 12D4 were unsuccessful, because its limited solubility made it difficult to prepare crystals. Reverting Ser to Pro at position 51 and extending the C- terminus with the EIDK sequence that is present in human fibronectin improved the solubility. This monobody, termed 12D5 (FIG.3A), had four-fold weaker affinity to KRAS(G12D)•GTPγS 171580388v1than 12D4, but still maintained high specificity to KRAS(G12D) over KRAS(WT) and slight preference to KRAS(G12D)•GTPγS over KRAS(G12D)•GDP (FIG.10A). The crystal structure of 12D5 in complex with KRAS(G12D) was determined. Two copies of the complex were observed in the asymmetric unit, but, intriguingly, with different states of the nucleotide bound to KRAS(G12D), one copy with GTPγS and the other with GDP. The conformations and the Mg2+coordination of GTPγS and GDP are consistent with those in other KRAS structures (FIG. 10B). In the 12D5-KRAS(G12D)•GTPγS complex, the N-terminal region of the symmetry- related KRAS(G12D) molecule is engaged in coordinating Mg2+(FIG.10C). The presence of GDP in the other copy is likely due to the hydrolysis of GTPγS used for crystallization, as in the 12D1-KRAS(G12D)•GDP structure.

[0159] The overall conformations of the two 12D5-KRAS complexes in the asymmetric unit as well as that of the 12D1-KRAS complex are nearly identical except for the switch I region (FIGS.4A, 4B, 10C), which is consistent with the ability of 12D5 to bind to both nucleotide states of KRAS(G12D) and the small differences in the sequences between 12D1 and 12D5 (FIG.3A).

[0160] A conspicuous difference between the interfaces in the GTPγS- and GDP-bound complexes is the interactions involving S80Mb. It directly interacts with GTPγS, whereas it has no interaction with GDP (FIG.4C), rationalizing the preferential binding of 12D5 toward KRAS(G12D)•GTP over KRAS(G12D)•GDP. In addition, switch I displayed distinct conformations, including partial disorder, between the GTPγS- and GDP-bound complexes (FIGS.4A, 10C), indicating that the switch I region is inherently flexible.

[0161] Although two residues in the FG loop are mutated in 12D5 (W78F and Y79W) with respect to 12D1, these mutations do not alter the FG loop conformation, and key interactions involving the common residues between 12D1 and 12D5, including that between G81Mband G12DRAS, are maintained (FIG.4C). A mutated position in 12D5, W79Mb, presents its side chain more deeply into the S-II pocket than Y79Mbin 12D1 (FIG.4D). The interface area between 12D5 and KRAS(G12D) is indeed larger than that between 12D1 and KRAS(G12D) (943 Å2vs 914 Å2; see Table 2, infra). Thus, a reasoned explanation for this behavior is that the W78F and Y79W mutations in 12D3, 12D4 and 12D5 increased the binding surface area while keeping the FG loop conformation, contributing to improved affinity compared with 12D1.

[0162] The conformations of the switch I and II regions in KRAS(G12D)•GTPγS in complex with 12D5 also substantially differ from that in the RAF RBD-bound RAS (FIG.11A). Given the high similarity between 12D5- and 12D1-bound KRAS(G12D) molecules (except for 171580388v1the switch I region), this observation rationalizes the inhibition by 12D1 of the interaction between the RAF RBD and KRAS(G12D) (FIG.1D). MRTX1133 causes similar but smaller- scale distortion of the RBD-binding surface of KRAS(G12D)•GppCp and disrupts the RBD– KRAS(G12D) interaction (Hallin et al., “The KRAS(G12C) Inhibitor MRTX849 Provides Insight toward Therapeutic Susceptibility of KRAS-Mutant Cancers in Mouse Models and Patients,” Cancer Discov 10:54-71 (2020), which is hereby incorporated by reference in its entirety). Similar incompatibility was found between the conformations of 12D5-bound KRAS(G12D)•GTPγS and KRAS bound to the RBD of PI3K (FIG.11B), indicating that these monobodies also inhibit KRAS(G12D)-mediated activation of PI3K. In addition, structural overlay showed that 12D5 binding to RAS would interfere with the interaction of RAS and SOS (FIG.11C), confirming that 12D1 and its variants inhibit allosteric activation of SOS by KRAS(G12D)•GTP. Example 6 - Deep Mutational Scanning

[0163] To comprehensively define residues of 12D4 important for binding to KRAS(G12D), deep mutational scanning (Fowler, “High-resolution Mapping of Protein Sequence-function Relationships,” Nat Methods 7:741-746 (2010), which is hereby incorporated by reference in its entirety) was performed. Using the crystal structures as a guide, a total of 19 residues in 12D4 were selected for mutation based on the expectation that these residues are located in or near the binding interface. For each of these 19 residues, a yeast-display library was generated in which one of these positions at a time was diversified to all 20 amino acids (FIG.5A). The library was sorted into three classes: first, clones that exhibited a binding profile similar to the wild-type 12D4 when measured with 10 nM KRAS(G12D) (“high binders”); second, those similar to the wild type when measured with 100 nM KRAS(G12D) (“binders”); and third, those that did not show binding when measured with 100 nM KRAS(G12D) (“nonbinders”) (FIG.5A). The experiment was performed with KRAS(G12D)•GTPγS and KRAS(G12D)•GDP, resulting in a total of six pools. These pools were subjected to deep sequencing and the numbers of reads for individual mutations were determined. The following were identified: 181 and 160 unique mutants for binders and nonbinders to KRAS(G12D)•GTPγS, and 149 and 186 unique mutants for binders and nonbinders to KRAS(G12D)•GDP. In total, 362 unique sequences were generated. As expected, there were high degrees of overlap between the high binder and binder pools, and there was little overlap between the nonbinder pool and the high binder and binder pools, indicating successful separation of clones with distinct binding properties (FIGS.5B, 5C). 171580388v1

[0164] In both high binder and binder pools, only two of the 19 positions, K75Mband G81Mb, did not permit any substitution. As described above, the backbone NH group of G81Mbis directly engaged with the Asp12RASside chain in the crystal structure of 12D1. The inability to mutate G81Mbstrongly indicates the importance of this interaction, as the side chain alterations are likely to perturb the position and orientation of the interaction between G81MbNH and Asp12RAS. The side chain of K75Mbalso points toward the side chain carboxyl of Asp12RAS. Intriguingly, the positively charged Nε of K75Mbdoes not directly interact with the Asp12RAScarboxyl. Instead, it is cradled by three backbone carbonyl groups of monobody residues (residues 76Mb, 79Mband 81Mb) (FIG.5D), indicating that this feature is important for maintaining the ring-like structure of the FG loop that supports most of the monobody residues interacting with the S-II pocket (FIGS.2B, 4C).

[0165] An additional eight positions allowed limited numbers of substitutions in the high binder and binder pools. Residues 76-79 and 82 in the FG loop fall in this class. This segment is tightly packed in the S-II pocket (FIGS.4C, 4D), rationalizing these characteristics. High conservation of the remaining residues in this class can also be rationalized by the crystal structure. V30Mband F31Mbform direct hydrophobic contacts with the α3 Helix (FIG.5E), V29Mbis part of the hydrophobic core of the monobody scaffold and important for its stability (Koide et al., “High-affinity Single-domain Binding Proteins with a Binary-code Interface,” Proc Natl Acad Sci USA 104:6632-6637 (2007), which is hereby incorporated by reference in its entirety). G52Mbforms a tight turn with a positive phi angle, an energetically reasonable conformation only for Gly. F31 can be replaced with His, Lys, Leu and Try, substations that may maintain close ring stacking or hydrophobic packing against H95 (FIG.5E). Similar ring stacking is observed in a KRAS-specific DARPin, K13 (FIG.5F) (Bery et al., “KRAS-specific Inhibition Using a DARPin Binding to a Site in the Allosteric Lobe,” Nat Commun 10:2607 (2019), which is hereby incorporated by reference in its entirety). The remaining positions allowed most substitutions. Together, the deep mutational scanning data are consistent with the 12D1 and 12D5 crystal structures, further validating them.

[0166] A few positions show different patterns between the corresponding pools recovered with KRAS(G12D)•GTPγS and KRAS(G12D)•GDP, (residues 27Mb, 51Mb, 77Mb, 79Mband 82Mb; FIGS.5B, 5C), indicating that these residues sense differences between the GTP- and GDP-bound states. However, minimal conformational differences of these residues and their adjacent residues were noted in the 12D5 crystal structures (FIGS.4B, 4C), indicating that additional information is required for rationalizing these data. Still, these data may guide future 171580388v1efforts in expanding the sequences of KRAS(G12D)-binding monobodies and tuning their nucleotide-state specificity.

[0167] Although depicted in the figures, the mutational scanning results are also presented in Tables A and B, infra. VSSVPTKLEVVAATPTSLLVSWDAPAX27XXXXYX33ITYGETGGNSPVQX47FX49V X51XXKSTATISGLKPGVDYTITVYAX75XXXXXXXSSPISINYRT (SEQ ID NO: 24) Each of the above-identified X residues can independently be wild-type (as identified in the tables below) or one of the recited mutations (as identified in Tables A and B, infra). Also contemplated are combinations of any two or more, any three or more, any four or more, any five or more, any six or more, any seven or more, any eight or more, any nine or more, any ten or more, any eleven or more, any twelve or more, any thirteen or more, any fourteen or more, any fifteen or more, any sixteen or more, any seventeen or more, any eighteen or more, or all nineteen of the X residues bearing one of the non-wildtype mutations. Example 7 - Intracellularly Expressed Monobodies Selectively Engage KRAS(G12D)

[0168] Monobodies 12D2, 12D3 and 12D4 were reformatted in the form of mCherry fusion proteins into genetically encoded reagents and expressed them intracellularly. These monobodies colocalized with overexpressed KRAS(G12D) fused to EGFP, but not with EGFP- KRAS(WT) (FIG.6A), indicating that these monobodies selectively engaged with KRAS(G12D) in the cellular context. Discussion of Examples 1-7

[0169] Monobodies developed in this work further expand the toolbox of biologic reagents for controlling oncogenic RAS mutants and elucidating RAS biology. While MRTX1133 achieves extraordinarily high specificity and potency toward KRAS(G12D), the preceding examples demonstrate success in developing a biologics reagent with what is believed to be the highest level of specificity to date toward KRAS(G12D). The favorable attributes of monobody reagents, including the ease of genetic encoding and intracellular delivery, flexibility in constructing fusion proteins including those with E3 ligase subunits, e.g., VHL and SPOP, and with fluorescent proteins, and the ability to control their expression in a temporal and tissue- specific manner, will complement small molecule inhibitors in advancing RAS research (Akkapeddi et al., “Monobodies as Tool Biologics for Accelerating Target Validation and 171580388v1Druggable Site Discovery,” RSC Med Chem 12:1839-1853 (2021), which is hereby incorporated by reference in its entirety).

[0170] The crystal structures of 12D1 and 12D5 in complex with KRAS(G12D) show remarkable similarity with the structure of MRTX1133 bound to KRAS(G12D), in particular, the placement of a nitrogen atom that interacts with the side chain of Asp12RAS, a feature that appears to be crucial for achieving high specificity toward the G12D mutation. At the same time, the 12D1 and 12D5 structures add snapshots of the S-II pocket to the growing body of atomic structures of RAS bound to noncovalent S-II-pocket ligands (Sakamoto et al. “Generation of KS- 58 as the First K-Ras(G12D)-inhibitory Peptide Presenting Anti-cancer Activity in vivo,” Sci Rep 10:21671 (2020); Zhang et al., “GTP-State-Selective Cyclic Peptide Ligands of K-Ras(G12D) Block Its Interaction with Raf,” ACS Cent Sci 6:1753-1761 (2020); Wang et al., “Identification of MRTX1133, a Noncovalent, Potent, and Selective KRAS(G12D) Inhibitor,” J Med Chem 65:3123-3133 (2022), each of which is hereby incorporated by reference in its entirety), which collectively define a highly malleable nature of this pocket. The deep mutational scanning data consisting of hundreds of 12D4 variants substantially expand the knowledge of the structure- activity relationship of ligands binding to the S-II pocket. Together, these data inform further design of inhibitors for KRAS(G12D).

[0171] KRAS(G12D)-specific monobodies were developed using standard protein- engineering technologies. This approach is in direct contrast to the challenge that small molecule inhibitor discovery for KRAS(G12D) has presented over the past four decades. The discovery of the S-II pocket with the covalent inhibitors for KRAS(G12C) was crucial for revising the notion that RAS has no pockets suitable for small molecules and for achieving the “initial traction” toward the ultimate development of MRTX1133 (Wang et al., “Identification of MRTX1133, a Noncovalent, Potent, and Selective KRAS(G12D) Inhibitor,” J Med Chem 65:3123-3133 (2022), which is hereby incorporated by reference in its entirety). By contrast, the 12D1 monobody was developed using a combination of positive and negative sorting of monobody libraries without prior knowledge of a targetable pocket. These results demonstrate the maturity of synthetic binding protein technologies to achieve exquisite specificity and potency even for the most challenging targets. Furthermore, advances in technologies for cellular delivery of proteins and nucleic acids enable therapeutic use of monobodies targeting intracellular molecules. 171580388v1Table 2: X-ray diffraction data collection and refinement statisticsData Collection Space group P 6422 P 21221 Cell dimensions a, b, c (Å) 156.48, 156.48, 64.62 151.39, 44.73, 88.16 α, β, γ (˚) 90, 90, 120 90, 90, 90 Resolution (Å) 50-2.52 (2.56-2.52)a50-2.44 (2.48-2.44)aRmerge0.092 (0.324) 0.091 (0.308) Rpim0.033 (0.113) 0.061 (0.207) CC1 / 2 0.986 (0.973) 0.939 (0.905) CC* 0.996 (0.993) 0.984 (0.975) │I σ │20.3 (7.4) 12.4 (5.0)Completeness (%) 99.9 (99.9) 98.4 (99.7) Redundancy 8.7 (9.0) 3.3 (3.2) Refinement Resolution (Å) 45.17-2.52 42.90-2.44 No. reflections 16,122 23,331 Rwork / Rfree0.171 / 0.213 0.201 / 0.245 No. atoms Protein 2034 4019 Ligand / Ion 30 86 Water 144 102 B-factors Protein 48.43 48.88 Ligand / Ion 33.51 50.81 Water 46.41 41.25 R.m.s. deviations Bond lengths (Å) 0.008 0.005 Bond angles (˚) 1.26 1.05 Ramachandran favored (%) 98.43 97.60 Ramachandran allowed (%) 1.57 2.20 Ramachandran outliers (%) 0.00 0.20 Clashscore 4.66 5.31 Interface area (Å2)b 914.4946.2 (GTPγS)Monobody-KRAS(G12D) 942.9 (GDP) a Values in parentheses are for highest-resolution shell. b Interface area calculation was performed using PDBePISA server from the EBI / EMBL. 171580388v1Table A: Binding to KRAS(G12D)•GTPγS171580388v1Table B: Binding to KRAS(G12D)•GDP

[0172] Although preferred embodiments have been depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be made without departing from the spirit of the invention and these are therefore considered to be within the scope of the invention as defined in the claims which follow. 171580388v1

Claims

WHAT IS CLAIMED IS:

1. A mutant KRAS binding polypeptide, said binding polypeptide comprising a fibronectin type III (FN3) domain having one or more modified loop sequences comprising a modified FG loop amino acid sequence, a modified BC loop amino acid sequence, a modified DE loop amino acid sequence, or a combination thereof, and optionally one or more modified beta strand sequences, wherein said one or more modified loop sequences and said one or more optional modified beta strand sequences enable selective binding to a mutant KRAS comprising an G12D substitution at residue 12 of KRAS (KRASG12D) over wildtype KRASG12.

2. The mutant KRAS binding polypeptide of claim 1, wherein said binding polypeptide binds to mutant KRASG12Din an active GTP-bound state or in the GDP-bound state.

3. The mutant KRAS binding polypeptide of claim 1 or claim 2, wherein said binding polypeptide selectively binds to mutant KRASG12Dover a mutant KRAS comprising a G12C substitution at residue 12 of KRAS (KRASG12C) or a mutant KRAS comprising a G12V substitution at residue 12 of KRAS (KRASG12V).

4. The binding polypeptide of any one of claims 1–3, wherein the modified FG loop amino acid sequence comprises SEQ ID NO:

6.

5. The binding polypeptide of any one of claims 1–4, wherein the modified FG loop amino acid sequence is selected from any one of SEQ ID NOs: 2-5.

6. The binding polypeptide of any one of claims 1–5, wherein the modified BC loop amino acid sequence comprises SEQ ID NO:

17.

7. The binding polypeptide of claim 1–6, wherein the modified BC loop amino acid sequence is selected from any one of SEQ ID NOs: 13-16.

8. The binding polypeptide of any one of claims 1–5, wherein the modified DE loop amino acid sequence comprises SEQ ID NO:

11.

9. The binding polypeptide of any one of claims 1–8, wherein the modified DE loop amino acid sequence is selected from any one of SEQ ID NOs: 7-10. 171580388v110. The binding polypeptide of any one of claims 1–9, wherein the FN3 domain is a human fibronectin type III tenth domain (10Fn3) of SEQ ID NO:1 comprising the at least one modified loop amino acid sequence.

11. The binding polypeptide of claim 10, wherein the one or more modified beta strands in the10Fn3 domain comprises an amino acid substitution in one of the C, D, F, and G beta-strands, or a combination thereof.

12. The binding polypeptide of claim 11, wherein the amino acid substitution is at one or more residues selected from Y31 and R33 of SEQ ID NO:

1.

13. The binding polypeptide of claim 12, wherein the amino acid substitution at Y31 is Y31F, Y31H, Y31K, or Y31L.

14. The binding polypeptide of claim 12, wherein the amino acid substitution at R33 is R33D, R33A, R33C, R33E, R33G, R33H, R33I, R33K, R33L, R33M, R33N, R33Q, R33S, R33T, R33V, R33W, or R33Y.

15. The binding polypeptide of claim 11 or 12, wherein the amino acid substitution is at one or more residues selected from E47 and T49 of SEQ ID NO:

1.

16. The binding polypeptide of claim 15, wherein the amino acid substitution at E47 is E47A, E47C, E47D, E47F, E47G, E47H, E47I, E47K, E47L, E47M, E47N, E47P, E47Q, E47R, E47S, E47T, E47V, E47W, or E47Y.

17. The binding polypeptide of claim 15, wherein the amino acid substitution at T49 is T49A, T49H, T49I, T49K, T49P, T49Q, T49R, T49S, T49V.

18. The binding polypeptide of any one of claims 1–17, wherein the FN3 domain comprises an FG loop amino acid sequence of SEQ ID NO: 6, a BC loop amino acid sequence of SEQ ID NO: 17, and a DE loop amino acid sequence of SEQ ID NO: 11 (Consensus).

19. The binding polypeptide of any one of claims 1-18, wherein the FN3 domain comprises: (i) an FG loop amino acid sequence of SEQ ID NO: 2, a BC loop amino acid sequence of SEQ ID NO: 13, and a DE loop amino acid sequence of SEQ ID NO: 7 (12D1); or 171580388v1(ii) an FG loop amino acid sequence of SEQ ID NO: 3, a BC loop amino acid sequence of SEQ ID NO: 14, and a DE loop amino acid sequence of SEQ ID NO: 8 (12D2); or (iii) an FG loop amino acid sequence of SEQ ID NO: 4, a BC loop amino acid sequence of SEQ ID NO: 15, and a DE loop amino acid sequence of SEQ ID NO: 9 (12D3); or (iv) an FG loop amino acid sequence of SEQ ID NO: 5, a BC loop amino acid sequence of SEQ ID NO: 16, and a DE loop amino acid sequence of SEQ ID NO: 10 (12D4); or (v) an FG loop amino acid sequence of SEQ ID NO: 5, a BC loop amino acid sequence of SEQ ID NO: 13, and a DE loop amino acid sequence of SEQ ID NO: 7 (12D5).

20. The binding polypeptide of any one of claims 1-18, wherein the FN3 domain comprises a C-terminal extension sequence of SEQ ID NO:

22.

21. The binding polypeptide of any one of claims 1–20, wherein the FN3 domain comprises an amino acid sequence that is at least 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 18–21 and 23.

22. The binding polypeptide of any one of claims 1–20, wherein the FN3 domain comprises an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 18–21 and 23.

23. The binding polypeptide of any one of claims 1–20, wherein the FN3 domain comprises an amino acid sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 18–21 and 23.

24. The binding polypeptide of any one of claims 1–20, wherein the FN3 domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 18-21 and 23.

25. The binding polypeptide of any one of claims 1–20, wherein the FN3 domain comprises a variant of the amino acid sequence of SEQ ID NO: 21 that includes one or more of the substitutions identified in Table A or Table B.

26. The binding polypeptide of any one of claims 1–20, further comprising a detectable label. 171580388v127. The binding polypeptide of claim 26, wherein the detectable label is selected from the group of paramagnetic ions, radioisotopes, fluorescent dyes, NMR-detectable substances, and X-ray imaging agents.

28. A mutant KRAS binding polypeptide conjugate, said conjugate comprising: a first portion, said first portion comprising the mutant KRAS binding polypeptide of any one of claims 1-25; and a second portion coupled to said first portion, said second portion selected from the group consisting of an E3 ubiquitin ligase ligand, an E3 ligase subunit, an E2 ubiquitin- conjugating enzyme, or an E2 recruiter.

29. The mutant KRAS binding polypeptide conjugate of claim 28, wherein the second portion of the conjugate comprises an E3 ubiquitin ligase ligand.

30. The mutant KRAS binding polypeptide conjugate of claim 29, wherein the E3 ubiquitin ligase ligand is a small molecule ligand for an E3 ubiquitin ligase selected from the group consisting of cereblon, Von Hippel-Lindau (VHL), Inhibitor of Apoptosis (IAP), MDM2, RNF114, DCAF16, DCAF15, Kelch-like ECH-associated protein 1 (KEAP1), FEM1B, and aryl hydrocarbon receptor.

31. The mutant KRAS binding polypeptide conjugate of claim 28, wherein the E3 ligase subunit is selected from the group consisting of SPOP (speckle type POZ protein), E3 ubiquitin-protein ligase CHIP, Ankyrin repeat and SOCS box protein 1 (ASB1), Suppressor of cytokine signaling 2 (SOCS2), DNA damage-binding protein 2 (DDB2), Protein cereblon (CRBN), von Hippel-Lindau disease tumor suppressor (VHL), S-phase kinase-associated protein 2 (SKP2), F-box / WD repeat-containing protein 7 (FBW7), and F-box / WD repeat-containing protein 1A isoform 2.

32. The mutant KRAS binding polypeptide conjugate of claim 28, wherein the second portion of the conjugate comprises an E2 ubiquitin-conjugating enzyme or an E2 recruiter.

33. The mutant KRAS binding polypeptide conjugate of claim 28 further comprising a delivery vehicle linked to one or both of the first and second portions. 171580388v134. The mutant KRAS binding polypeptide conjugate of claim 33, wherein the delivery vehicle comprises a toxin.

35. The mutant KRAS binding polypeptide conjugate of claim 34, wherein the toxin is an attenuated toxin.

36. The mutant KRAS binding polypeptide conjugate of claim 35, wherein the attenuated toxin is an attenuated anthrax toxin, an attenuated diphtheria toxin, attenuated Shiga and Shiga-like toxin.

37. The mutant KRAS binding polypeptide conjugate of claim 33, wherein the delivery vehicle comprises an antibody or binding fragment thereof that binds a cell surface molecule.

38. The mutant KRAS binding polypeptide conjugate of claim 37, wherein the cell surface molecule is a cancer cell specific surface molecule selected from CUB domain- containing protein 1 (CDCP1), Intercellular adhesion molecule 1 (ICAM1), Integrin beta-5 (ITGB5), (5'-nucleotidase ) NT5E, Tumor necrosis factor receptor superfamily member 3 (LTBR), Complement decay-accelerating factor (CD55), Aminopeptidase N (ANPEP), CD79, Trophoblast glycoprotein (TPBG), Integrin beta-1 (ITGB1), Prostaglandin F2 receptor negative regulator (PTGFRN), Integrin alpha-5 (ITGA5), and Exosome complex protein LRP1 (LRP1).

39. The mutant KRAS binding polypeptide conjugate of claim 33, wherein the delivery vehicle is a cell penetrating ligand.

40. The mutant KRAS binding polypeptide conjugate of claim 39, wherein the cell penetrating ligand is selected from TAT, cR10, Pep-1, penetratin, and transportan.

41. The mutant KRAS binding polypeptide conjugate of claim 33, wherein the delivery vehicle is PEI-polypeptide conjugate that is chemically linked to the mutant KRASG12Dbinding polypeptide or mutant KRASG12Dbinding peptide conjugate.

42. The mutant KRAS binding polypeptide conjugate of claim 41, wherein the PEI-polypeptide conjugate comprises a PEI-albumin, a PEI-fibrinogen, a PEI-globulin, or a PEI- DARPin. 171580388v143. The mutant KRAS binding polypeptide conjugate of claim 28, wherein the conjugate is linked to or encapsulated by a delivery vehicle selected from a nanoparticle, a polymer-based particle, and a lipid-based particle.

44. A polynucleotide encoding the mutant KRAS binding polypeptide of any one of claims 1-25 or the mutant KRAS binding polypeptide conjugate of claim 28.

45. The polynucleotide of claim 43, wherein said polynucleotide is an mRNA encoding the mutant KRAS binding polypeptide of any one of claims 1–25.

46. A vector comprising the isolated polynucleotide of claim 44 or claim 45.

47. The vector of claim 46, wherein said vector is a viral vector.

48. The vector of claim 47, wherein said viral vector is selected from the group consisting of lentiviral vector, an adeno-associated viral vector, vaccinia vector, and a retroviral vector.

49. A delivery vehicle comprising the polynucleotide of claim 44 or claim 45 or the vector of any one of claims 46–48.

50. The delivery vehicle of claim 49, wherein the delivery vehicle is selected from a nanoparticle, a polymer-based particle, and a lipid-based particle.

51. A fusion construct comprising: a first portion, said first portion comprising a polynucleotide encoding the mutant KRAS binding polypeptide of any one of claims 1-25; and a second portion, operatively coupled to said first portion, said second portion comprising a polynucleotide encoding a E3 ligase subunit or an E2 ubiquitin-conjugating enzyme, or an imaging protein or peptide thereof.

52. The fusion construction of claim 51, wherein the second portion is a polynucleotide encoding an E3 ligase subunit.

53. The fusion construct of claim 52, wherein the polynucleotide encodes an E3 ligase subunit selected from the group consisting of: SPOP (speckle type POZ protein), E3 ubiquitin-protein ligase CHIP, Ankyrin repeat and SOCS box protein 1 (ASB1), Suppressor of cytokine signaling 2 (SOCS2), DNA damage-binding protein 2 (DDB2), Protein cereblon 171580388v1(CRBN), von Hippel-Lindau disease tumor suppressor (VHL), S-phase kinase-associated protein 2 (SKP2), F-box / WD repeat-containing protein 7 (FBW7), and F-box / WD repeat-containing protein 1A isoform 2.

54. The fusion construction of claim 51, wherein the second portion is a polynucleotide encoding an E2 ubiquitin-conjugating enzyme.

55. The fusion construct of claim 51, wherein the second portion is a polynucleotide encoding an imaging protein or peptide thereof.

56. The fusion construct of claim 55, wherein the imaging protein or peptide is selected from a Aequorea green fluorescent protein and derivatives thereof, Anthozoan fluorescent protein and derivatives thereof, Discosoma red fluorescent protein and derivatives thereof, Anemonia fluorescent protein and derivatives thereof, Firefly luciferase protein and derivatives thereof, Renilla luciferase protein and derivatives thereof, and bacterial luciferase protein and derivatives thereof.

57. A pharmaceutical composition comprising the mutant KRAS binding polypeptide of any one of claims 1–27, the mutant KRAS binding polypeptide conjugate of any one of claims 28–43, the isolated polynucleotide of any one of claims 44–45, the vector of any one of claims 46–48, or the fusion construct of any one claims 49-56, and a pharmaceutical carrier.

58. A method for treating cancer in a subject, said method comprising: administering, to the subject having cancer, the pharmaceutical composition of claim 57 in an amount effective to treat the cancer.

59. The method of claim 58, wherein the cancer is characterized by cancerous cells having an oncogenic mutation in KRAS.

60. The method of claim 59, wherein the mutation in KRAS is a KRASG12Dmutation.

61. The method of any one of claims 58-60, wherein the cancer is pancreatic cancer, lung cancer, breast cancer, colon cancer, glioma, solid tumor, melanoma, glioblastoma multiforme, leukemia, renal cell carcinoma, hepatocellular carcinoma, prostate cancer, and myeloma. 171580388v162. The method of any one of claims 58-60, wherein the polynucleotide is administered.

63. The method of any one of claims 58-60, wherein the mutant KRAS binding polypeptide or the mutant KRAS binding polypeptide conjugate is administered.

64. The method of any one of claims 58-63, wherein said administering is carried out orally, intravenously, intradermally, subcutaneously, intramuscularly, intraperitoneally, intra-arterially, buccally, intranasally, via nebulization, via bronchial inhalation, or intratumorally.

65. A method for causing cancer cell death, said method comprising: introducing the mutant KRAS binding polypeptide of any one of claims 1–27 or the mutant KRAS binding polypeptide conjugate of any one of claims 28–43 to a cancer cell under conditions to cause cancer cell death.

66. The method according to claim 65, wherein the cancer cell is in vitro.

67. The method according to claim 65, wherein the cancer cell is in vivo.

68. The method according to claim 65, wherein the cancer cell is characterized by having an oncogenic mutation in KRAS.

69. The method of claim 68, wherein the mutation in KRAS is a KRASG12Dmutation.

70. The method of any one of claims 65–69, wherein the cancer cell is a pancreatic cancer cell, lung cancer cell, breast cancer cell, colon cancer cell, glioma cell, solid tumor cell, melanoma cell, glioblastoma multiforme cell, renal carcinoma cell, hepatocellular carcinoma cell, prostate cancer cell, myeloma (plasma) cancer cell, and leukemia cancer cell.

71. The method according to claim 65, wherein said introducing is carried out by expressing a transgene encoding the mutant KRAS binding polypeptide or the mutant KRAS binding polypeptide conjugate in the cancer cell.

72. The method according to claim 71, wherein the transgene is introduced into the cancer cell. 171580388v173. The method according to claim 65, wherein said introducing is carried out by causing the cancer cell to uptake the mutant KRAS binding polypeptide or the mutant KRAS binding polypeptide conjugate.

74. A method for detecting the presence of KRAS(G12D) in a sample, the method comprising: contacting a sample with the mutant KRAS binding polypeptide according to any one of claims 1-27, and determining whether the mutant KRAS binding polypeptide specifically binds to KRAS(G12D).

75. The method according to claim 74, wherein the mutant KRAS binding polypeptide comprises a detectable label.

76. The method according to claim 74, wherein the sample is selected from a tissue section or specimen, a homogeneous tissue extract, a biological fluid, or a secretion.

77. A method for in vivo diagnostic imaging, the method comprising: administering a composition comprising the mutant KRAS binding polypeptide according to claim 26 under conditions effective to allow the mutant KRAS binding polypeptide to bind specifically to KRAS(G12D); and obtaining an image of a site in a patient where the mutant KRAS binding polypeptide has bound to KRAS(G12D).

78. The method according to claim 77, wherein said obtaining an image is carried out using computed tomography, positron emission tomography, magnetic resonance imaging, and single-photon emission computed tomography.

79. A method for screening compounds for binding to KRAS(G12D), the method comprising: exposing KRAS(G12D) to the mutant KRAS binding polypeptide according to any one of claims 1-27 and a compound being screened for binding activity; and determining whether the mutant KRAS binding polypeptide binds to KRAS(G12D) or the compound being screened binds to KRAS(G12D).

80. The method according to claim 79, wherein the mutant KRAS binding polypeptide comprises a detectable label, and said determining comprises measuring presence or absence of the label present following said exposing. 171580388v181. The method according to claim 79, wherein the amount of label present after said exposing is quantified.

82. The method according to claim 79, wherein said exposing is carried out with a plurality of compounds. 171580388v1

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