Forward-looking Statement This presentation contains forward-looking statements as defined in the Private Securities Litigation Reform Act of 1995 regarding, among other things, the potential regulatory path forward for

Corporate Presentation January 2023

Forward-looking Statement This

presentation contains forward-looking statements as defined in the Private Securities Litigation Reform Act of 1995 regarding, among other things, the potential regulatory path forward for tebipenem HBr and the potential approval of tebipenem HBr by

the U.S. Food and Drug Administration (FDA) and the timing thereof; the potential commercialization of tebipenem HBr and its future value, the potential receipt of milestone payments, and royalties on future sales of tebipenem HBr under the

GlaxoSmithKline Intellectual Property (No. 3) Limited (GSK) license agreement; Spero's cash runway and estimated cash balance; the future development and commercialization of SPR206 and SPR720; the potential number of patients who could be treated

by tebipenem HBr and SPR720 and market demand for tebipenem HBr and SPR720 generally; the effectiveness of tebipenem HBr and its potential impact on healthcare resource utilizations; expected broad access across payer channels for tebipenem HBr; the

anticipated shift in treating patients from intravenous to oral administration; the initiation, timing, progress and results of the Company's preclinical studies and clinical trials and its research and development programs, including

management's assessment of such results; the direct and indirect impact of the pandemic caused by an outbreak of a strain of coronavirus (COVID-19) on the Company's business and operations; the timing of the availability of data from the

Company's clinical trials; the timing of the Company's filings with regulatory agencies; product candidate benefits; competitive position; business strategies; objectives of management; potential growth opportunities; potential

market size; reimbursement matters; possible or assumed future results of operations; projected costs; and the Company's cash forecast and the availability of additional non-dilutive funding from governmental agencies beyond any initially

funded awards. In some cases, forward-looking statements can be identified by terms such as "may," "will," "should," "expect," "plan," "aim," "anticipate,"

"could," "intent," "target," "project," "contemplate," "believe," "estimate," "predict," "potential" or "continue" or the

negative of these terms or other similar expressions. All statements other than statements of historical facts contained in this presentation are forward-looking statements. The Company may not actually achieve the plans, intentions or

expectations disclosed in these forward-looking statements. Actual results or events could differ materially from the plans, intentions and expectations disclosed in these forward-looking statements as a result of various factors, including

whether the FDA will ultimately approve tebipenem HBr and, if so, the timing of any such approval; whether the FDA will require any additional clinical data or place labeling restrictions on the use of tebipenem HBr that would add costs for the

Company, delay approval and/or reduce the commercial prospects of tebipenem HBr; the Company's need for additional funding; the lengthy, expensive, and uncertain process of clinical drug development; the Company's reliance on third

parties to manufacture, develop, and commercialize its product candidates, if approved; the ability to develop and commercialize the Company's product candidates, if approved; the potential impact of the COVID-19 pandemic; the Company's

ability to retain key personnel; whether results obtained in preclinical studies and clinical trials will be indicative of results obtained in future clinical trials and whether preliminary data from the Company's clinical trials will be

predictive of final results from such trials; whether the Company's product candidates will advance through the preclinical development and clinical trial process on a timely basis, or at all, taking into account such factors as the

effects of possible regulatory delays, slower than anticipated patient enrollment, manufacturing challenges, clinical trial design, clinical data requirements and clinical outcomes; whether the results of such clinical trials will warrant submission

for approval from the FDA or equivalent foreign regulatory agencies; decisions made by the FDA and equivalent foreign regulatory agencies with respect to the development and commercialization of the Company's product

candidates; the commercial potential of the Company's product candidates; the Company's ability to obtain adequate third-party reimbursement for its product candidates; whether the Company will satisfy all of the pre-conditions

to receipt of the milestone payments under its various license and collaboration agreements; whether BARDA elects to exercise its second option under the Company's agreement with BARDA; the Company's ability to implement its strategic

plans; the Company's ability to obtain, maintain and enforce intellectual property and other proprietary rights for its product candidates; the risks and uncertainties related to market conditions; whether the Company's cash

resources will be sufficient to fund its continuing operations for the periods and/or trials anticipated; and other factors discussed in the "Risk Factors" section of the Company's periodic reports filed with the U.S. Securities

and Exchange Commission (SEC), and risks described in other filings the Company may make with the SEC in the future. The forward-looking statements included in this presentation represent the Company's views as of the date of this

presentation. The Company anticipates that subsequent events and developments will cause its views to change. However, while the Company may elect to update these forward-looking statements at some point in the future, it specifically disclaims any

obligation to do so. These forward-looking statements should not be relied upon as representing the Company's views as of any date subsequent to the date of this presentation.

Spero Therapeutics, Inc. A Leader in

Infectious and Rare Diseases SPR720: Potential Novel, First Line, Oral Therapy for NTM Infections Strong Balance Sheet Supported by World Class Partnerships for Tebipenem HBr, SPR206 NTM = non-tuberculous mycobacterial; MDR = multidrug

resistant infections; Tebipenem HBr = tebipenem pivoxil hydrobromide (formerly SPR994), 1. CHEST Foundation, About Nontuberculous Mycobacteria (NTM) lung infections; Kevin L. Winthrop et al., Incidence and Prevalence of Nontuberculous Mycobacterial

Lung Disease in a Large United States Managed Care Health Plan, 2008-2015 (Feb. 2020) 2. As of Dec 31, 2022, This figure is unaudited, and considered a preliminary estimate that is subject to change Three late-stage medicines, targeting indications

with high unmet need and strong commercial prospects Seasoned executive team, with significant clinical, regulatory and operational experience (10+ credited drug approvals at prior companies) NTM: Large orphan disease patient population

(approximately 240k patients worldwide1). No approved first line therapies Oral therapy targeting bacterial DNA replication Initiated Phase 2 proof-of-concept study in 4Q 2022 Phase 1 SAD/MAD study and in vivo efficacy data support

potential for NTM patients $600M global partnership with GSK fully supports tebipenem HBr clinical program through potential approval Partnership milestones, non-dilutive funding, and over $100 million2 in available cash provides operating

Late-Stage Pipeline Addressing Major

Unmet Needs NTM: Nontuberculous mycobacteria; MDR: Multi-drug resistant; BAL: Bronchoalveolar lavage; cUTI: Complicated urinary tract infection Spero Retains World-Wide Rights Spero Licensed World-Wide Rights to GSK Spero Retains Rights in United

Spero Strategy: Invest in diseases and

settings of care supportive of blockbuster growth Non-DRG reimbursement High unmet need with strong economic benefit $1 B+ Sales* *Estimated Peak Year Worldwide Sales *Trademarks are properties of their respective owners

NTM: Absence of Effective and

Well-tolerated Drugs Leaves Patients Without Options SPR720 has orphan drug designation and, if approved, could be the only novel first line oral treatment for NTM Infection Colonization Symptoms (progressive lung injury) Treatment No

well-tolerated, effective treatments Refractory Limited opportunity For clinical benefit Non-tuberculous mycobacterial disease (NTM) is a chronic infection with debilitating pulmonary symptoms. Insmed's Arikayce is the only approved therapy,

although in refractory NTM-MAC. Patients need therapy early in their disease journey. No other approved therapies exist. Source: The voice of the Patient; Non-Tuberculous Mycobacterial (NTM) Lung Infection Public Meeting: October 15,

2015 Report Date: April 2016

SPR720: Novel, First Line Oral

Candidate Designed to Treat NTM Infections Broad spectrum, oral candidate: applicable to both first line and refractory patients Approximately 95,000 patients in US. Total of 245,000 in US, Europe* and Japan1 More than 75% of NTM patients are

non-refractory1; lack any approved options to treat NTM SPR720 has potency against range of NTM pathogens, including MAC and M. Abcessus2 Once daily dose supported by clinical and non-clinical studies Supportive safety/ tolerability data Selected

500 - 1000mg once daily dose range for Phase 2 supported by concordant in vivo and in vitro PK/PD models BAL study in non-human primates supports lung exposure; macrophage data shows intracellular and extracellular activity Data at 500 - 1000mg once

daily in Phase 1 SAD/MAD studies supportive of advancement to Phase 2 clinical studies 1. CHEST Foundation, About Nontuberculous Mycobacteria (NTM) lung infections; Kevin L. Winthrop et al., Incidence and Prevalence of Nontuberculous Mycobacterial

Lung Disease in a Large United States Managed Care Health Plan, 2008-2015 (Feb. 2020); Wall Street research reports; *Europe refers to Germany, United Kingdom, Italy, Netherlands and France. 2. Citation Brown-Elliott BA, Rubio A, Wallace RJ, Jr.

2018. In vitro susceptibility testing of a novel benzimidazole, SPR719, against nontuberculous mycobacteria. Antimicrobial Agents Chemotherapy 62:e01503-18

Robust in vitro data supporting

advancement of the program SPR719 exhibits potent in vitro activity against a range of NTM species NTM Species N SPR719 MIC90 Amikacin MIC90 M. avium complex 41 2 32 M. abscessus subsp. abscessus 30 4 16 M. abscessus subsp. massiliense 10 2 16 M.

chelonae 10 4 32 M. fortuitum group 10 1 1 M. immunogenum 10 8 16 M. simiae 10 2 16 AUC = area under the drug concentration-time curve; MIC = minimum inhibitory concentration; MIC = minimum inhibitory concentration; MIC90 = MIC that inhibits

90% of the isolates; NTM = nontuberculous mycobacteria SPR719 is the active moiety of the prodrug SPR720 and is used for in vitro testing. Brown-Elliott BA, et al. Antimicrob Agents Chemother 2018; 62:e01503-18. SPR720 Investigator Brochure Edition

5; 07Apr2022. Hollow fiber infection model revealed a high intracellular penetration into infected macrophages

SPR720 Pulmonary Activity versus M.

avium ATCC 700898 in a Murine Chronic Infection Model ++ - +++ ++ + + + + - - - + + - + SPR720 Monotherapy CLR +/- EMB +/-SPR720 CLR + RFB +/- EMB +/-SPR720 D1 D27 D60 SPR720 (mg/kg/day) 10 QD 30 QD 100 BID 100 QD CLR CLR 72030 CLR EMB CLR EMB 72030

CLR RFB CLR RFB 72030 CLR RFB EMB CLR RFB EMB 72030 Untreated SPR720 as monotherapy and in combination with SOC agents SPR720 monotherapy: Dose responsive reduction in bacterial burden versus the untreated control SPR720 at 30 mg/kg/day improved the

activity of CLR and CLR + EMB CLR: clarithromycin EMB: ethambutol Verma, Deepshikha & Peterson, Chelsea & Cotroneo, Nicole & Stokes, Suzanne & Ordway, Diane. (2020). 1637. SPR720, A Novel Benzamidazole Gyrase Inhibitor, Demonstrates

Potent Efficacy Against Mycobacterium avium ATCC 700898 in a Chronic C3HeBFeJ Mouse Infection Model. Open Forum Infectious Diseases. Untreated

Phase 1: Favorable Tolerability at

Doses with Predicted Therapeutic Activity Phase 1 results show SPR720 was well-tolerated with exposures above predicted therapeutic levels Single Ascending Dose Cohorts (N = 56) Multiple Ascending Dose Cohorts (N = 40) No Serious Adverse Events

(SAE) reported; Mild GI symptoms at 1500mg Dose dependent increase in plasma exposure observed No clinically significant differences in safety or PK between healthy elderly and healthy non-elderly subjects 500mg and 1000mg doses evaluated

over 7- and 14-day dose ranges No SAEs reported; Most common adverse events (AE) among all cohorts was mild diarrhea not requiring discontinuation of therapy No clinically significant lab findings: ALT levels >1.5x ULN noted in 3 subjects;

maximum values <3x ULN and rapidly reversible. No Hy's law cases One discontinuation at 1500mg: pancreatic enzyme elevation that was asymptomatic, monitorable and reversible Talley, Angela et al. "1288. Phase 1 First-in-Human Single-

and Multiple-Ascending Dose Trial Demonstrates Pharmacokinetics (PK) and Tolerability of SPR720, an Oral DNA GyrB Inhibitor for Mycobacterial Infections." Open Forum Infectious Diseases vol. 7,Suppl 1 S659. 31 Dec. 2020,

doi:10.1093/ofid/ofaa439.1471

SPR720 Patient Focus: First line

NTM-MAC 75% of NTM patients are treatment na ve or treatment inexperienced, and eligible for first line therapy Treatment Na ve Bronchial hygiene/clearance, exercise, education Treatment Inexperienced NTM Refractory Disease None

approved; Generic antibiotic regimens: (clarithromycin, ethambutol, rifampin) Inhaled Arikayce approved for NTM (Refractory pulmonary MAC) Five-year NTM-PD mortality rate: 35% SPR720 Target: First Line 1. Ku JH, Henkle E,

Carlson KF, Marino M, Brode SK, Marras TK, Winthrop KL. Evaluation of Mycobacterium avium complex pulmonary disease treatment completion and adherence to ATS/IDSA guidelines. Clin Infect Dis. 2022 May 24. 2. Wallace RJ Jr, Brown-Elliott BA, McNulty

S, Philley JV, Killingley J, Wilson RW, York DS, Shepherd S, Griffith DE. Macrolide/Azalide therapy for nodular/bronchiectatic mycobacterium avium complex lung disease. Chest. 2014. 3. Aug;Novosad SA, Henkle E, Schafer S, Hedberg K, Ku J, Siegel

SAR, Choi D, Slatore CG, Winthrop KL. Mortality after Respiratory Isolation of Nontuberculous Mycobacteria. A Comparison of Patients Who Did and Did Not Meet Disease Criteria. Ann Am Thorac Soc. 2017 Jul. 2. 2. Standard of Care Patient

Challenges Respiratory symptoms, fatigue, impact daily living, quality of life Only 50% go on to Rx therapy: Intolerability Ineffectiveness 75% of patients discontinue SoC after 1 year of treatment, due to tolerability,

ineffectivness 50% relapse rate even after therapy Reduced opportunity to improve how patients feels & functions, in setting of chronic lung damage

Study SPR720-202 (NCT05496374)

designed to achieve early and robust proof of concept Study SPR720-202 (NCT05496374): Phase 2a study in NTM-PD N SPR719 MIC50 A Study to Evaluate the Efficacy, Safety, Tolerability, and Pharmacokinetics of SPR720, as Compared With Placebo for

the Treatment of Participants With Mycobacterium avium Complex (MAC) Pulmonary Disease 30 2 Treatment Population Four Treatment Arms Objectives Treatment-na ve/-inexperienced patients with NTM due to M. avium complex Approximately 35

patients Placebo, n=9 SPR720 (500 mg), n=9 SPR720 (1000 mg), n=9 SPR720 (1000 mg), n=4 Evaluate: 1. Safety & Tolerability 2. Plasma PK 3.

Microbiological response 4. How patients feel & function Key goal of this Phase 2a study is to show a signal of efficacy in patients, measuring both clinical and microbiological outcomes 27 patients randomized 1:1:1 (blinded)

Intense PK substudy (open label) MAC = M. avium complex; NTM-PD = nontuberculous mycobacterial pulmonary disease; PK = pharmacokinetics Next Steps for the Development Plan Initiated clinical sites, patient screening began in Q4 2022 Potential

to be first to show early microbiological response as stand-alone agent versus placebo

Oral Carbapenem Tebipenem

Large Market Opportunity for

Patients Able to Be Treated at Home Targeted patients often cycle through multiple therapies 7.9 M UTI Patients in the US 2.7 M Market 780 K IV Carbapenem Pip/tazo 1.9 M >2nd line Oral (Resistant/ Failed) Lack of effective oral treatment options

has resulted in increased Outpatient visits Emergency department visits Unwarranted outpatient IV use Unnecessary hospitalizations Hospital days Home Health and Long-Term Care (LTC) stays post-hospitalization Tebipenem Focus 2.7M UTI

prescriptions 2nd line + Oral or IV therapy Tebipenem HBr: Qualified Infectious Disease Product (QIDP) exclusivity Intellectual Property (IP) runway until 2038 Source: IQVIA 2019; Komodo 2020 data; Becton Dickinson Market Research, 2020 Pip/tazo =

Piperacillin and tazobactam 5.2 M 1 st line Oral 1.6 M 2 nd line Oral (Resistant/Failed) 1.1 M IV Therapy/3rd+ line Oral (Resistant/Failed)

Clinical Experience Supports Safety,

Efficacy of Tebipenem1 Tebipenem pivoxil evaluated in 24 trials enrolling over 2500 subjects Approved in Japan since 2009 Spero experience: Global cUTI Phase 3, enrolling 1372 subjects Meiji Seika experience 741 Adult subjects

evaluated, across 17 efficacy and pharmacology trials 440 pediatric subjects evaluated, across 6 efficacy and pharmacology trials Tebipenem pivoxil was generally well tolerated, comparable to common, approved oral beta lactam antibiotics and IV

carbapenems Tebipenem pivoxil met its primary endpoint in 3 double blind placebo-controlled efficacy trials in pediatric pneumonia, otitis media, and sinusitis Approved for pediatric pneumonia, otitis media, sinusitis, over 4

million patients dosed to date Extensive post-marketing safety and efficacy surveillance completed, covering 3,331 patients No issues of safety were observed, and adequate efficacy was demonstrated 1. Akash Jain, Luke Utley,

Thomas R. Parr, Thomas Zabawa & Michael J. Pucci (2018): Tebipenem, the first oral carbapenem antibiotic, Expert Review of Anti-infective Therapy, DOI: 10.1080/14787210.2018.1496821

Results from Spero's ADAPT-PO

Phase 3 Clinical Trial Published in The New England Journal of Medicine Results from Spero's Phase 3 ADAPT-PO study, as recently published in The New England Journal of Medicine (NEJM), demonstrated that oral tebipenem HBr was well-tolerated

and non-inferior to IV ertapenem, based on its trial protocol, in the treatment of 1372 enrolled adult patients with cUTI or acute pyelonephritis.

Exclusive License Agreement with GSK

for Tebipenem HBr and Equity Investment Global Collaboration Financial Terms GSK received exclusive license to develop and commercialize tebipenem pivoxil and tebipenem HBr in all territories, except Japan and certain other Asian countries (Meiji